Pyrrolobenzodiazepines

ABSTRACT

A compound with the formula I, wherein R 2  is of formula II, where A is a C 5-7  aryl group, X is selected from the group consisting of: OH, SH, CO 2 H, COH, N═C═O, NHNH 2 , CONHNH 2 , (III), (IV), and NHR N , wherein R N  is selected from H and C 1-4  alkyl, and either: (i) Q 1  is a single bond, and Q 2  is selected from a single bond and —Z—(CH 2 ) n —, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or (ii) Q 1  is —CH═CH—, and Q 2  is a single bond; and its conjugates.

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position on one monomer unit, and an exo double bond at the C2 position on the other monomer unit.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:

They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.

It has been previously disclosed that the biological activity of this molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimer is SG2000 (SJG-136):

(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).

Dimeric PBD compounds bearing C2 aryl substituents, such as SG2202 (ZC-207), are disclosed in WO 2005/085251:

and in WO2006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents (Howard, P. W., et al., Bioorg. Med. Chem. (2009), doi: 10.1016/j.bmc1.2009.09.012).

Due to the manner in which these highly potent compounds act in cross-linking DNA, these molecules have been made symmetrically. This provides for straightforward synthesis, either by constructing the PBD moieties simultaneously having already formed the dimer linkage, or by reacting already constructed PBD moieties with the dimer linking group.

WO 2010/043880 and WO 2011/130613 disclose dimeric PBDs similar to those above which are not symmetrical due to a group on one of the C2 aryl groups which can be joined to a linker to a cell binding agent, such as an antibody.

WO 2011/130616 discloses similar PBD dimer conjugates, where the PBD monomer which is not linked to the cell binding agent has a double bond between C2 and C3 but a non-aromatic C2 substituent.

WO 2013/053873 discloses PBD dimer conjugates, where the PBD monomer linked to the cell binding agent has a double bond between C2 and C3 and is linked via a propenyl group, and the PBD monomer which is not linked to the cell binding agent has a double bond between C2 and C3 and an aromatic or non-aromatic C2 substituent.

DISCLOSURE OF THE INVENTION

In the PBD monomer which is not linked to the cell binding agent, the C ring may be prone to become fully aromatic by oxidation (‘aromatization’). This process may be spontaneous, or the aromatization may occur during synthesis. For example, sibiromycin:

is known to aromatise, which leads to inactivity of the PBD. Aromatization can be detected using NMR (by determining the present of an addition aromatic hydrogen).

To overcome this issue, the present inventors have synthesised PBD dimers where the C-ring not linked to the cell binding agent has an exo-double bond, thus retaining the sp² centre at C2.

The present invention comprises a compound with the formula I:

wherein: R² is of formula II′:

where A is a C₅₋₇ aryl group, X is selected from the group consisting of: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

and NHR^(N), wherein R^(N) is selected from H and C₁₋₄ alkyl, and either: (i) Q¹ is a single bond, and Q² is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or (ii) Q¹ is —CH═CH—, and Q² is a single bond; R¹² is selected from ═CH₂, ═CHR^(D1) and ═CR^(D1)R^(D2), where R^(D1) and R^(D2) are independently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Sn and halo; where R and R′ are independently selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups; either: (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl; (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R″ is a C₃₋₁₂ alkylene group, which chain is optionally interrupted by one or more heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ and R⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, wherein if R¹¹ and R^(11′) are SO_(z)M, M may represent a divalent pharmaceutically acceptable cation.

A second aspect of the present invention provides the use of a compound of the first aspect of the invention in the manufacture of a medicament for treating a proliferative disease. The second aspect also provides a compound of the first aspect of the invention for use in the treatment of a proliferative disease.

One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.

A third aspect of the present invention comprises a compound of formula II:

wherein: R² is of formula II′:

where A is a C₅₋₇ aryl group, X is selected from the group comprising: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

NHR^(N), wherein R^(N) is selected from the group comprising H and C₁₋₄ alkyl, and either: (i) Q¹ is a single bond, and Q² is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or (ii) Q¹ is —CH═CH—, and Q² is a single bond; R¹² is selected from ═CH₂, ═CHR^(D1) and ═CR^(D1)R^(D2), where R^(D1) and R^(D2) are independently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; where R and R′ are independently selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups; R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Sn and halo; either: (a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹ is O-Prot^(O), wherein Prot^(O) is an oxygen protecting group; or (b) R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxo group; R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ and R⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹.

A fourth aspect of the present invention comprises a method of making a compound of formula I from a compound of formula II by deprotection of the imine bond.

A fifth aspect of the present invention provides a method of making a compound of the first or third aspect of the invention, comprising at least one of the method steps set out below.

In a sixth aspect, the present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein a PBD is a dimer of formula I (supra).

In some embodiments, the Conjugates have the following formula III:

L-(LU-D)_(p)  (III)

wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit comprising a PBD dimer according to formula I, wherein LU is connected to D via the X substituent of R². The subscript p is an integer of from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.

The PBD dimer D is of formula I, except that X is selected from the group comprising: O, S, C(═O), C═, NH(C═O), NHNH, CONHNH,

NR^(N), wherein R^(N) is selected from the group comprising H and C₁₋₄ alkyl.

In a seventh aspect of the invention, there is provided a drug linker having formula IV:

DLU-D  (IV)

wherein DLU is a Drug Linker unit, and D is a Drug unit comprising a PBD dimer according to formula I, wherein DLU is connected to D via the X substituent of R².

BRIEF DESCRIPTION OF FIGURE

FIG. 1 shows the effect of a conjugate of the invention on a tumour.

DEFINITIONS Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca²⁺ and Mg²⁺. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

-   -   saturated monocyclic hydrocarbon compounds:         cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),         cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),         dimethylcyclopropane (C₅), methylcyclobutane (C₅),         dimethylcyclobutane (C₆), methylcyclopentane (C₆),         dimethylcyclopentane (C₇) and methylcyclohexane (C₇);     -   unsaturated monocyclic hydrocarbon compounds:         cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),         cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene         (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),         methylcyclopentene (C₆), dimethylcyclopentene (C₇) and         methylcyclohexene (C₇); and     -   saturated polycyclic hydrocarbon compounds:         norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₆), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇); O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆); N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂O₁: oxadiazine (C₆); O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀), acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆); O₁: furan (oxole) (C₅); S₁: thiophene (thiole) (C₅); N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆); N₂O₁: oxadiazole (furazan) (C₅); N₃O₁: oxatriazole (C₅); N₁S₁: thiazole (C₅), isothiazole (C₅); N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅), pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆; N₃: triazole (C₅), triazine (C₆); and, N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are not limited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),         isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine         (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,         adenine, guanine), benzimidazole (N₂), indazole (N₂),         benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),         benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),         benzothiazole (N₁S₁), benzothiadiazole (N₂S);     -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene         (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline         (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),         benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),         quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),         naphthyridine (N₂), pteridine (N₄);     -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);     -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),         dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),         perimidine (N₂), pyridoindole (N₂); and,     -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene         (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),         phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),         thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),         phenazine (N₂).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I. Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkoxy group, discussed below), a C₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxy group), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxy group), preferably a C₁₋₇ alkyl group. Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkyl group. Examples of C₁₋₇ alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy). Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in the case of a “cyclic” acetal group, R¹ and R², taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt). Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt). Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂, —C(Et)(OEt)₂, and —C(Et)(OMe)(OEt). Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt). Oxo (keto, -one): ═O. Thione (thioketone): ═S. Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, =NEt, and ═NPh. Formyl (carbaldehyde, carboxaldehyde): —C(═O)H. Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl), a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl), or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably a C₁₋₇ alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃ (t-butyryl), and —C(═O)Ph (benzoyl, phenone). Carboxy (carboxylic acid): —C(═O)OH. Thiocarboxy (thiocarboxylic acid): —C(═S)SH. Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH. Thionocarboxy (thionocarboxylic acid): —C(═S)OH. Imidic acid: —C(═NH)OH. Hydroxamic acid: —C(═NOH)OH. Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh. Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph. Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃, and —OC(═O)OPh. Amino: —NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case of a “cyclic” amino group, R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), or tertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³). Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and —C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃, —C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R² is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂, —OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂. Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂, —NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and =NEt. Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂, and —C(═NMe)NMe₂.

Nitro: —NO₂. Nitroso: —NO. Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC. Cyanato: —OCN. Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN. Isothiocyano (isothiocyanato): —NCS. Sulfhydryl (thiol, mercapto): —SH. Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthio group), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are not limited to, —SCH₃ and —SCH₂CH₃. Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyl disulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are not limited to, —SSCH₃ and —SSCH₂CH₃. Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH₃ and —S(═O)CH₂CH₃. Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, including, for example, a fluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉ (nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl). Sulfinic acid (sulfino): —S(═O)OH, —SO₂H. Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H. Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH₃ (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate). Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)₂OCH₃ (methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl; ethyl sulfonate). Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃. Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)₂CH₃ (mesylate) and —OS(═O)₂CH₂CH₃ (esylate). Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃. Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh. Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂, —S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh. Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH. Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅. Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅. Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphino groups include, but are not limited to, —PH₂, —P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀ aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂. Phosphonic acid (phosphono): —P(═O)(OH)₂. Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and —P(═O)(OPh)₂. Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂. Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and —OP(═O)(OPh)₂. Phosphorous acid: —OP(OH)₂. Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂. Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramidite substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂, —OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂. Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidate substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂, —OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are not limited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example, —CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂— (pentylene) and —CH₂CH₂CH₂CH-₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but are not limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and —CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ alkenylene and alkynylene groups) include, but are not limited to, —C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂ cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate).

Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Conjugates

The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker Unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L¹), and a Spacer unit (L²). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.

In one aspect, such a Conjugate is shown below in formula IIla:

L-(A ¹ _(a)-L ¹ _(s)-L ² _(y)-D)_(p)  (IIIa)

-   -   wherein:     -   L is the Ligand unit; and     -   -A¹ _(a)-L¹ _(s)-L² _(y)-is a Linker unit (LU), wherein:     -   -A¹-is a Stretcher unit,     -   a is 1 or 2,     -   L¹-is a Specificity unit,     -   s is an integer ranging from 1 to 12,     -   -L²-is a Spacer unit,     -   y is 0, 1 or 2;     -   -D is an PBD dimer; and     -   p is from 1 to 20.

In some embodiments of this aspect, a is 1, s is 1 and y is 0, such that the conjugate is of formula IIIa-1:

L-(A ¹-L ¹-D)_(p)  (IIIa-1)

In another aspect, such a Conjugate is shown below in formula IIIb:

Also illustrated as:

L-(A ¹ _(a)-L ² _(y)(-L ¹ _(s))-D)_(p)  (IIIb)

-   -   wherein:     -   L is the Ligand unit; and     -   -A¹ _(a)-L¹ _(s)(L² _(y))- is a Linker unit (LU), wherein:     -   -A¹-is a Stretcher unit linked to a Spacer unit (L²),     -   a is 1 or 2,     -   L¹-is a Specificity unit linked to a Spacer unit (L²),     -   s is an integer ranging from 0 to 12,     -   -L²-is a Spacer unit,     -   y is 0, 1 or 2;     -   -D is a PBD dimer; and     -   p is from 1 to 20.

Preferences

The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

In one embodiment, the Conjugate has the formula:

L-(A ¹ _(a)-L ¹ _(s)-L ² _(y)-D)_(p)

-   -   wherein L, A¹, a, L¹, s, L², D and p are as described above.

In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell.

An exemplary formula for the conjugate is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug         unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity         unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding         Agent, L² is a Spacer unit, which is a covalent bond, a         self-immolative group or together with —OC(═O)— forms a         self-immolative group, and L² is optional.

Another exemplary formula is illustrated below:

CBA-A ¹ _(a)-L ¹ _(s)-L ² _(y)-*

-   -   where the asterisk indicates the point of attachment to the Drug         unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity         unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding         Agent, L² is a Spacer unit which is a covalent bond or a         self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is         0 or 1 or 2.

A further exemplary formula is illustrated below:

CBA-A ¹-L ¹-*

where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit and A¹ is a Stretcher unit connecting L¹ to the Cell Binding Agent.

In the embodiments illustrated above, L¹ can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L², when a self-immolative group(s) is present. When the Specificity unit L¹ is cleaved, or the linkage (i.e., the covalent bond) between L¹ and L² is cleaved, the self-immolative group releases the Drug unit (D).

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug         (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit         connected to L², A¹ is a Stretcher unit connecting L² to the         Cell Binding Agent, L² is a self-immolative group, and a is 1 or         2, s is 1 or 2, and y is 1 or 2.

In the various embodiments discussed herein, the nature of L¹ and L² can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L¹ is cleavable, the structure and/or sequence of L¹ is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L¹ units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L¹ units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.

In some embodiments, L¹ may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme.

In one embodiment, L¹ is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L¹ may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L² is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group.

In one embodiment, where L¹ is cleavable by the action of an enzyme and L² is present, the enzyme cleaves the bond between L¹ and L², whereby the self-immolative group(s) release the Drug unit.

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,     -   —C(═O)O—,     -   —NHC(═O)—,     -   —OC(═O)—,     -   —OC(═O)O—,     -   —NHC(═O)O—,     -   —OC(═O)NH—,     -   —NHC(═O)NH, and     -   —O— (a glycosidic bond).

An amino group of L¹ that connects to L² may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.

A hydroxy group of L¹ that connects to L² may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the Drug         unit, the wavy line indicates the point of attachment to the L¹,         Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The         phenylene ring is optionally substituted with one, two or three         substituents as described herein.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC).

The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0):

-   -   where the asterisk indicates the attachment to the Drug, L* is         the activated form of the remaining portion of the linker and         the released Drug unit is not shown. These groups have the         advantage of separating the site of activation from the Drug.

In another embodiment, —C(═O)O— and L² together form a group selected from:

-   -   where the asterisk, the wavy line, Y, and n are as defined         above. Each phenylene ring is optionally substituted with one,         two or three substituents as described herein. In one         embodiment, the phenylene ring having the Y substituent is         optionally substituted and the phenylene ring not having the Y         substituent is unsubstituted.

In another embodiment, —C(═O)O— and L² together form a group selected from:

-   -   where the asterisk, the wavy line, Y, and n are as defined         above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or         CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the covalent bond between L¹ and L² is a cathepsin labile (e.g., cleavable) bond.

In one embodiment, L¹ comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selected from:

-   -   -Phe-Lys-,     -   -Val-Ala-,     -   -Val-Lys-,     -   -Ala-Lys-,     -   -Val-Cit-,     -   -Phe-Cit-,     -   -Leu-Cit-,     -   -Ile-Cit-,     -   -Phe-Arg-, and     -   -Trp-Cit-;         where Cit is citrulline. In such a dipeptide, —NH— is the amino         group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selected from:

-   -   -Phe-Lys-,     -   -Val-Ala-,     -   -Val-Lys-,     -   -Ala-Lys-, and     -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,     -   -Pro-Pro-, and     -   -Val-Glu-.

Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference.

In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;     -   Asn: Trt, Xan;     -   Asp: Bzl, t-Bu;     -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;     -   Glu: Bzl, t-Bu;     -   Gln: Trt, Xan;     -   His: Boc, Dnp, Tos, Trt;     -   Lys: Boc, Z—Cl, Fmoc, Z;     -   Ser: Bzl, TBDMS, TBDPS;     -   Thr: Bz;     -   Trp: Boc;     -   Tyr: Bzl, Z, Z—Br.

In one embodiment, —X₂— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L² is present.

In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X₂—.

Where a self-immolative group is present, —X₂— is connected directly to the self-immolative group. In one embodiment, —X₂— is connected to the group Y of the self-immolative group. Preferably the group —X₂—CO— is connected to Y, where Y is NH.

—X₁— is connected directly to A¹. In one embodiment, —X₁— is connected directly to A¹. Preferably the group NH—X₁— (the amino terminus of X₁) is connected to A¹. A¹ may comprise the functionality —CO— thereby to form an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the group —X₁—X₂-PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug         unit, and the wavy line indicates the point of attachment to the         remaining portion of L¹ or the point of attachment to A¹.         Preferably, the wavy line indicates the point of attachment to         A¹.

Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In another embodiment, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug         unit, the wavy line indicates the point of attachment to A¹, Y         is a covalent bond or a functional group, and E is a group that         is susceptible to cleavage thereby to activate a self-immolative         group.

E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO₂ or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase.

The group Y may be a covalent bond.

The group Y may be a functional group selected from:

-   -   —C(═O)—     -   —NH—     -   —O—     -   —C(═O)NH—,     -   —C(═O)O—,     -   —NHC(═O)—,     -   —OC(═O)—,     -   —OC(═O)O—,     -   —NHC(═O)O—,     -   —OC(═O)NH—,     -   —NHC(═O)NH—,     -   —NHC(═O)NH,     -   —C(═O)NHC(═O)—,     -   SO₂, and     -   —S—.

The group Y is preferably —NH—, —CH₂—, —O—, and —S—.

In some embodiments, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug         unit, the wavy line indicates the point of attachment to A, Y is         a covalent bond or a functional group and E is glucuronic acid         (e.g., β-glucuronic acid). Y is preferably a functional group         selected from —NH—.

In some embodiments, L¹ and L² together represent:

-   -   where the asterisk indicates the point of attachment to the         remainder of L² or the Drug unit, the wavy line indicates the         point of attachment to A¹, Y is a covalent bond or a functional         group and E is glucuronic acid (e.g., β-glucuronic acid). Y is         preferably a functional group selected from —NH—, —CH₂—, —O—,         and —S—.

In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A¹. In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit.

The Specificity unit L¹ and the Ligand unit are indirectly connected via the Stretcher unit.

L¹ and A¹ may be connected by a bond selected from:

-   -   —C(═O)NH—,     -   —C(═O)O—,     -   —NHC(═O)—,     -   —OC(═O)—,     -   —OC(═O)O—,     -   —NHC(═O)O—,     -   —OC(═O)NH—, and     -   —NHC(═O)NH—.

A¹ may be of formula:

-L ^(A)-A ²

wherein L^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene. and A² is selected from:

-   -   n is 0 to 6, e.g. 5;

-   -   n is 0 to 6, e.g. 5;

-   -   n is 0 or 1 (e.g. 1), and m is 0 to 30 (preferably 0 to 10, 1 to         8, 4 to 8, most preferably 4 or 8);

-   -   n is 0 or 1 (e.g. 1), and m is 0 to 30 (preferably 0 to 10, 1 to         8, 4 to 8, most preferably 4 or 8);         where the asterisk indicates the point of attachment to L¹, the         wavy line indicates the point of attachment to L^(A).

In a particular embodiment, L^(A) is (L^(A1-1)) and A² is

where n is 1, and m is 0 to 8, e.g. 8

In one embodiment, the Stretcher unit A¹ is present, the Specificity unit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L² is a bond.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)NH—,     -   —C(═O)O—,     -   —NHC(═O)—,     -   —OC(═O)—,     -   —OC(═O)O—,     -   —NHC(═O)O—,     -   —OC(═O)NH—, and     -   —NHC(═O)NH—.

In one embodiment, L¹ and D are preferably connected by a bond selected from:

-   -   —C(═O)NH—, and     -   —NHC(═O)—.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

In one embodiment, L¹-D is:

—NH—X₁—X₂—CO—NH—*

where —NH—X₁—X₂—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L¹ or the point of attachment to A¹. Preferably, the wavy line indicates the point of attachment to A¹.

In one embodiment, the dipeptide is valine-alanine and L¹-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalanine-lysine and L¹-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent.

In one embodiment, the Drug Linker unit has the formula:

-   -   where the asterisk indicates the point of attachment to the Drug         unit, G¹ is a Stretcher group (A¹) to form a connection to a         Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a         covalent bond or together with —OC(═O)— forms a self-immolative         group(s).

In another embodiment, the Drug Linker unit has the formula:

G ¹-L ¹-L ²-*

-   -   where the asterisk indicates the point of attachment to the Drug         unit, G¹ is a Stretcher unit (A¹) to form a connection to a         Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a         covalent bond or a self-immolative group(s).

In particular of these embodiments, the Drug Linker has the formula:

G ¹-L ¹-*

-   -   where the asterisk indicates the point of attachment to the Drug         unit, G¹ is a modified Stretcher unit (A¹) to form a connection         to a Ligand unit and L¹ is a Specificity unit.

L¹ and L² are as defined above. References to connection to A¹ can be construed here as referring to a connection to G¹.

The functional group G¹ forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.

G¹ may be of formula:

G ^(A)-A ²

where A² is as defined above and G^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene.

In a particular embodiment, G^(A) is (G^(A1-1)) and A² is

where n is 1, and m is 0 to 8, e.g. 8.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.

The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1×10⁷ M⁻¹, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.

Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that binds integrin α_(v)β₆. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cys polypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid residue. Furthermore, the polypeptide may have the sequence

NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, β, ε, γ, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibody or antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody wherein a portion of the variable region, preferably a portion substantially less than the intact human variable domain, has been substituted by the corresponding sequence from a non-human species and wherein the modified variable region is linked to at least another part of another protein, preferably the constant region of a human antibody. The expression “humanized antibodies” includes human antibodies in which one or more complementarity determining region (“CDR”) amino acid residues and/or one or more framework region (“FW” or “FR”) amino acid residues are substituted by amino acid residues from analogous sites in rodent or other non-human antibodies. The expression “humanized antibody” also includes an immunoglobulin amino acid sequence variant or fragment thereof that comprises an FR having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Or, looked at another way, a humanized antibody is a human antibody that also contains selected sequences from non-human (e.g. murine) antibodies in place of the human sequences. A humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’, ‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as ‘veneering’), ‘composite antibodies’, ‘Human String Content Optimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are ‘grafted’ onto the human framework). In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues (this may happen when, for example, a particular FR residue has significant effect on antigen binding).

Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Thus, in general, a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a given non-human antibody specific for a particular epitope with a human V_(H) or V_(L) library and specific human V domains are selected against the antigen of interest. This selected human VH is then combined with a VL library to generate a completely human VH×VL combination. The method is described in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a human antibody are combined within the final antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations which limit or avoid human T cell epitopes in the final composite antibody V regions. Where required, T cell epitopes are limited or avoided by, exchanging V region segments contributing to or encoding a T cell epitope with alternative segments which avoid T cell epitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species) T-cell epitopes from the V regions of the therapeutic antibody (or other molecule). The therapeutic antibodies V-region sequence is analysed for the presence of MHC class II-binding motifs by, for example, comparison with databases of MHC-binding motifs (such as the “motifs” database hosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifs may be identified using computational threading methods such as those devised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutive overlapping peptides from the V-region sequences are testing for their binding energies to MHC class II proteins. This data can then be combined with information on other sequence features which relate to successfully presented peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.

Once potential second species (e.g. human) T-cell epitopes have been identified, they are eliminated by the alteration of one or more amino acids. The modified amino acids are usually within the T-cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and therefore, may not be adjacent in the primary structure). Most typically, the alteration is by way of substitution but, in some circumstances amino acid addition or deletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, so that the final molecule may be prepared by expression from a recombinant host using well established methods such as Site Directed Mutagenesis. However, the use of protein chemistry or any other means of molecular alteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable         region of the non-human (e.g. rodent) antibody (or fragment         thereof) by constructing a three-dimensional model of the         non-human antibody variable region;     -   (b) generating sequence alignments using relative accessibility         distributions from x-ray crystallographic structures of a         sufficient number of non-human and human antibody variable         region heavy and light chains to give a set of heavy and light         chain framework positions wherein the alignment positions are         identical in 98% of the sufficient number of non-human antibody         heavy and light chains;     -   (c) defining for the non-human antibody to be humanized, a set         of heavy and light chain surface exposed amino acid residues         using the set of framework positions generated in step (b);     -   (d) identifying from human antibody amino acid sequences a set         of heavy and light chain surface exposed amino acid residues         that is most closely identical to the set of surface exposed         amino acid residues defined in step (c), wherein the heavy and         light chain from the human antibody are or are not naturally         paired;     -   (e) substituting, in the amino acid sequence of the non-human         antibody to be humanized, the set of heavy and light chain         surface exposed amino acid residues defined in step (c) with the         set of heavy and light chain surface exposed amino acid residues         identified in step (d);     -   (f) constructing a three-dimensional model of the variable         region of the non-human antibody resulting from the substituting         specified in step (e);     -   (g) identifying, by comparing the three-dimensional models         constructed in steps (a) and (f), any amino acid residues from         the sets identified in steps (c) or (d), that are within 5         Angstroms of any atom of any residue of the complementarity         determining regions of the non-human antibody to be humanized;         and     -   (h) changing any residues identified in step (g) from the human         to the original non-human amino acid residue to thereby define a         non-human antibody humanizing set of surface exposed amino acid         residues; with the proviso that step (a) need not be conducted         first, but must be conducted prior to step (g).

Superhumanization

The method compares the non-human sequence with the functional human germline gene repertoire. Those human genes encoding canonical structures identical or closely related to the non-human sequences are selected. Those selected human genes with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these human FRs. This method is described in patent WO 2005/079479 A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with the repertoire of human germline genes and the differences are scored as Human String Content (HSC) that quantifies a sequence at the level of potential MHC/T-cell epitopes. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA pools encompassing all known heavy and light chain human germline gene frameworks. Humanised antibodies are then selected by e.g. panning of the phage displayed antibody library. This is described in Methods 36, 43-60 (2005).

Examples of cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodiments of the present invention are listed below.

Tumor-Associated Antigens and Cognate Antibodies

(1) BMPR1B (bone morphogenetic protein receptor-type IB)

Nucleotide

Genbank accession no. NM_001203 Genbank version no. NM_001203.2 GI:169790809 Genbank record update date: Sep. 23, 2012 02:06 PM

Polypeptide

Genbank accession no. NP_001194 Genbank version no. NP_001194.1 GI:4502431 Genbank record update date: Sep. 23, 2012 02:06 PM

CROSS-REFERENCES

ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004/063362 (Claim 2); WO2003/042661 (Claim 12); US2003/134790-A1 (Page 38-39); WO2002/102235 (Claim 13; Page 296); WO2003/055443 (Page 91-92); WO2002/99122 (Example 2; Page 528-530); WO2003/029421 (Claim 6); WO2003/024392 (Claim 2; FIG. 112); WO2002/98358 (Claim 1; Page 183); WO2002/54940 (Page 100-101); WO2002/59377 (Page 349-350); WO2002/30268 (Claim 27; Page 376); WO2001/48204 (Example; FIG. 4); NP_001194 bone morphogenetic protein receptor, type IB/pid=NP_001194.1.; MIM:603248; AY065994

(2) E16 (LAT1, SLC7A5) Nucleotide

Genbank accession no. NM_003486 Genbank version no. NM_003486.5 GI:71979931 Genbank record update date: Jun. 27, 2012 12:06 PM

Polypeptide

Genbank accession no. NP_003477 Genbank version no. NP_003477.4 GI:71979932 Genbank record update date: Jun. 27, 2012 12:06 PM

CROSS REFERENCES

Biochem. Biophys. Res.

Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267 (16):11267-11273); WO2004/048938 (Example 2); WO2004/032842 (Example IV); WO2003/042661 (Claim 12); WO2003/016475 (Claim 1); WO2002/78524 (Example 2); WO2002/99074 (Claim 19; Page 127-129); WO2002/86443 (Claim 27; Pages 222, 393); WO2003/003906 (Claim 10; Page 293); WO2002/64798 (Claim 33; Page 93-95); WO2000/14228 (Claim 5; Page 133-136); US2003/224454 (FIG. 3); WO2003/025138 (Claim 12; Page 150); NP_003477 solute carrier family 7 (cationic amino acid transporter, y+system), member 5/pid=NP_003477.3-Homo sapiens; MIM:600182; NM_015923.

(3) STEAP1 (Six Transmembrane Epithelial Antigen of Prostate) Nucleotide

Genbank accession no. NM_012449 Genbank version no. NM_012449.2 GI:22027487 Genbank record update date: Sep. 9, 2012 02:57 PM

Polypeptide

Genbank accession no. NP_036581 Genbank version no. NP_036581.1 GI:9558759 Genbank record update date: Sep. 9, 2012 02:57 PM

CROSS REFERENCES

Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004/065577 (Claim 6); WO2004/027049 (FIG. 1L); EP1394274 (Example 11); WO2004/016225 (Claim 2); WO2003/042661 (Claim 12); US2003/157089 (Example 5); US2003/185830 (Example 5); US2003/064397 (FIG. 2); WO2002/89747 (Example 5; Page 618-619); WO2003/022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG. 2A); six transmembrane epithelial antigen of the prostate; MIM:604415.

(4) 0772P (CA125, MUC16) Nucleotide

Genbank accession no. AF361486 Genbank version no. AF361486.3 GI:34501466 Genbank record update date: Mar. 11, 2010 07:56 AM

Polypeptide

Genbank accession no. AAK74120 Genbank version no. AAK74120.3 GI:34501467 Genbank record update date: Mar. 11, 2010 07:56 AM

CROSS REFERENCES

J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004/045553 (Claim 14); WO2002/92836 (Claim 6; FIG. 12); WO2002/83866 (Claim 15; Page 116-121); US2003/124140 (Example 16); GI:34501467;

(5) MPF (MPF, MSLN, SMR, Megakaryocyte Potentiating Factor, Mesothelin) Nucleotide

Genbank accession no. NM_005823 Genbank version no. NM_005823.5 GI:293651528 Genbank record update date: Sep. 2, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005814 Genbank version no. NP_005814.2 GI:53988378 Genbank record update date: Sep. 2, 2012 01:47 PM

CROSS REFERENCES

Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); WO2003/101283 (Claim 14); (WO2002/102235 (Claim 13; Page 287-288); WO2002/101075 (Claim 4; Page 308-309); WO2002/71928 (Page 320-321); WO94/10312 (Page 52-57); IM:601051.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (Sodium Phosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b) Nucleotide

Genbank accession no. NM_006424 Genbank version no. NM_006424.2 GI:110611905 Genbank record update date: Jul. 22, 2012 03:39 PM

Polypeptide

Genbank accession no. NP_006415 Genbank version no. NP_006415.2 GI:110611906 Genbank record update date: Jul. 22, 2012 03:39 PM

CROSS REFERENCES

J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004/022778 (Claim 2); EP1394274 (Example 11); WO2002/102235 (Claim 13; Page 326); EP0875569 (Claim 1; Page 17-19); WO2001/57188 (Claim 20; Page 329); WO2004/032842 (Example IV); WO2001/75177 (Claim 24; Page 139-140); MIM:604217.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, 25 Sema Domain, Seven Thrombospondin Repeats (Type 1 and Type 1-Like), Transmembrane Domain and Short Cytoplasmic Domain, (Semaphorin) 58) Nucleotide

Genbank accession no. AB040878 Genbank version no. AB040878.1 GI:7959148 Genbank record update date: Aug. 2, 2006 05:40 PM

Polypeptide

Genbank accession no. BAA95969 Genbank version no. BAA95969.1 GI:7959149 Genbank record update date: Aug. 2, 2006 05:40 PM

CROSS REFERENCES

Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004/000997 (Claim 1); WO2003/003984 (Claim 1); WO2002/06339 (Claim 1; Page 50); WO2001/88133 (Claim 1; Page 41-43, 48-58); WO2003/054152 (Claim 20); WO2003/101400 (Claim 11); Accession: Q9P283; Genew; HGNC:10737

(8) PSCA Hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene)

Nucleotide

Genbank accession no. AY358628 Genbank version no. AY358628.1 GI:37182377 Genbank record update date: Dec. 1, 2009 04:15 AM

Polypeptide

Genbank accession no. AAQ88991 Genbank version no. AAQ88991.1 GI:37182378 Genbank record update date: Dec. 1, 2009 04:15 AM

CROSS REFERENCES

Ross et al (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2); US2004/044180 (Claim 12); US2004/044179 (Claim 11); US2003/096961 (Claim 11); US2003/232056 (Example 5); WO2003/105758 16 (Claim 12); US2003/206918 (Example 5); EP1347046 (Claim 1); WO2003/025148 (Claim 20); GI:37182378.

(9) ETBR (Endothelin Type B Receptor) Nucleotide

Genbank accession no. AY275463 Genbank version no. AY275463.1 GI:30526094 Genbank record update date: Mar. 11, 2010 02:26 AM

Polypeptide

Genbank accession no. AAP32295 Genbank version no. AAP32295.1 GI:30526095 Genbank record update date: Mar. 11, 2010 02:26 AM

CROSS REFERENCES

Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002) Hum. Genet. 111, 198-206; WO2004/045516 (Claim 1); WO2004/048938 (Example 2); WO2004/040000 (Claim 151); WO2003/087768 (Claim 1); WO2003/016475 (Claim 1); WO2003/016475 (Claim 1); WO2002/61087 (FIG. 1); WO2003/016494 (FIG. 6); WO2003/025138 (Claim 12; Page 144); WO2001/98351 (Claim 1; Page 124-125); EP0522868 (Claim 8; FIG. 2); WO2001/77172 (Claim 1; Page 297-299); US2003/109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34); WO2004/001004.

(10) MSG783 (RNF124, Hypothetical Protein FLJ20315) Nucleotide

Genbank accession no. NM_017763 Genbank version no. NM_017763.4 GI:167830482 Genbank record update date: Jul. 22, 2012 12:34 AM

Polypeptide

Genbank accession no. NP_060233 Genbank version no. NP_060233.3 GI:56711322 Genbank record update date: Jul. 22, 2012 12:34 AM

CROSS REFERENCES

WO2003/104275 (Claim 1); WO2004/046342 (Example 2); WO2003/042661 (Claim 12); WO2003/083074 (Claim 14; Page 61); WO2003/018621 (Claim 1); WO2003/024392 (Claim 2; FIG. 93); WO2001/66689 (Example 6); LocusID:54894.

(11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate Cancer Associated Gene 1, Prostate Cancer Associated Protein 1, Six Transmembrane Epithelial Antigen of Prostate 2, Six Transmembrane Prostate Protein) Nucleotide

Genbank accession no. AF455138 Genbank version no. AF455138.1 GI:22655487 Genbank record update date: Mar. 11, 2010 01:54 AM

Polypeptide

Genbank accession no. AAN04080 Genbank version no. AAN04080.1 GI:22655488 Genbank record update date: Mar. 11, 2010 01:54 AM

CROSS REFERENCES

Lab. Invest. 82 (11):1573-1582 (2002)); WO2003/087306; US2003/064397 (Claim 1; FIG. 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962 (Claim 1; FIG. 4B); WO2003/104270 (Claim 11); WO2003/104270 (Claim 16); US2004/005598 (Claim 22); WO2003/042661 (Claim 12); US2003/060612 (Claim 12; FIG. 10); WO2002/26822 (Claim 23; FIG. 2); WO2002/16429 (Claim 12; FIG. 10); GI:22655488.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient Receptor Potential Cation Channel, Subfamily M, Member 4) Nucleotide

Genbank accession no. NM_017636 Genbank version no. NM_017636.3 GI:304766649 Genbank record update date: Jun. 29, 2012 11:27 AM

Polypeptide

Genbank accession no. NP_060106 Genbank version no. NP_060106.2 GI:21314671 Genbank record update date: Jun. 29, 2012 11:27 AM

CROSS REFERENCES

Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003)); US2003/143557 (Claim 4); WO2000/40614 (Claim 14; Page 100-103); WO2002/10382 (Claim 1; FIG. 9A); WO2003/042661 (Claim 12); WO2002/30268 (Claim 27; Page 391); US2003/219806 (Claim 4); WO2001/62794 (Claim 10 14; FIG. 1A-D); MIM:606936.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor)

Nucleotide

Genbank accession no. NM_003212 Genbank version no. NM_003212.3 GI:292494881 Genbank record update date: Sep. 23, 2012 02:27 PM

Polypeptide

Genbank accession no. NP_003203 Genbank version no. NP_003203.1 GI:4507425 Genbank record update date: Sep. 23, 2012 02:27 PM

CROSS REFERENCES

Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim 1); WO2003/083041 (Example 1); WO2003/034984 (Claim 12); WO2002/88170 (Claim 2; Page 52-53); WO2003/024392 (Claim 2; FIG. 58); WO2002/16413 (Claim 1; Page 94-95, 105); WO2002/22808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2); MIM:187395.

(14) CD21 (CR2 (Complement Receptor 2) or C3DR (C3d/Epstein Barr Virus Receptor) or Hs. 73792) Nucleotide

Genbank accession no M26004 Genbank version no. M26004.1 GI:181939 Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35786 Genbank version no. AAA35786.1 GI:181940 Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320; WO2004/045520 (Example 4); US2004/005538 (Example 1); WO2003/062401 (Claim 9); WO2004/045520 (Example 4); WO91/02536 (FIGS. 9.1-9.9); WO2004/020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79β, IGb (Immunoglobulin-Associated Beta), 829) Nucleotide

Genbank accession no NM_000626 Genbank version no. NM_000626.2 GI:90193589 Genbank record update date: Jun. 26, 2012 01:53 PM

Polypeptide

Genbank accession no. NP_000617 Genbank version no. NP_000617.1 GI:11038674 Genbank record update date: Jun. 26, 2012 01:53 PM

CROSS REFERENCES

Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004/016225 (Claim 2, FIG. 140); WO2003/087768, US2004/101874 (Claim 1, page 102); WO2003/062401 (Claim 9); WO2002/78524 (Example 2); US2002/150573 (Claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003/048202 (Claim 1, pages 306 and 309); WO 99/58658, U.S. Pat. No. 6,534,482 (Claim 13, FIG. 17A/B); WO2000/55351 (Claim 11, pages 1145-1146); MIM:147245

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 Domain Containing Phosphatase Anchor Protein 1a), SPAP1B, SPAP1C) Nucleotide

Genbank accession no NM_030764 Genbank version no. NM_030764.3 GI:227430280 Genbank record update date: Jun. 30, 2012 12:30 AM

Polypeptide

Genbank accession no. NP_110391 Genbank version no. NP_110391.2 GI:19923629 Genbank record update date: Jun. 30, 2012 12:30 AM

CROSS REFERENCES

AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004/016225 (Claim 2); WO2003/077836; WO2001/38490 (Claim 5; FIG. 18D-1-18D-2); WO2003/097803 (Claim 12); WO2003/089624 (Claim 25); MIM:606509.

(17) HER2 (ErbB2) Nucleotide

Genbank accession no M11730 Genbank version no. M11730.1 GI:183986 Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA75493 Genbank version no. AAA75493.1 GI:306840 Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-15 880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15, 426-429; WO2004/048938 (Example 2); WO2004/027049 (FIG. 11); WO2004/009622; WO2003/081210; WO2003/089904 (Claim 9); WO2003/016475 (Claim 1); US2003/118592; WO2003/008537 (Claim 1); WO2003/055439 (Claim 29; FIG. 1A-B); WO2003/025228 (Claim 37; FIG. 5C); WO2002/22636 (Example 13; Page 95-107); WO2002/12341 (Claim 68; FIG. 7); WO2002/13847 (Page 71-74); WO2002/14503 (Page 114-117); WO2001/53463 (Claim 2; Page 41-46); WO2001/41787 (Page 15); WO2000/44899 (Claim 52; FIG. 7); WO2000/20579 (Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004/043361 (Claim 7); WO2004/022709; WO2001/00244 (Example 3; FIG. 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1

Antibodies Abbott: US20110177095

-   -   For example, an antibody comprising CDRs having overall at least         80% sequence identity to CDRs having amino acid sequences of SEQ         ID NO:3 (CDR-H1), SEQ ID NO:4 (CDR-H2), SEQ ID NO:5 (CDR-H3),         SEQ ID NO:104 and/or SEQ ID NO:6 (CDR-L1), SEQ ID NO:7 (CDR-L2),         and SEQ ID NO:8 (CDR-L3), wherein the anti-HER2 antibody or         anti-HER2 binding fragment has reduced immunogenicity as         compared to an antibody having a VH of SEQ ID NO:1 and a VL of         SEQ ID NO:2.

Biogen: US20100119511

-   -   For example, ATCC accession numbers: PTA-10355, PTA-10356,         PTA-10357, PTA-10358     -   For example, a purified antibody molecule that binds to HER2         comprising a all six CDR's from an antibody selected from the         group consisting of BIIB71F10 (SEQ ID NOs:11, 13), BIIB69A09         (SEQ ID NOs:15, 17); BIIB67F10 (SEQ ID NOs:19, 21); BIIB67F11         (SEQ ID NOs:23, 25), B11B66A12 (SEQ ID NOs:27, 29), BIIB66C01         (SEQ ID NOs:31, 33), BIIB65C10 (SEQ ID NOs:35, 37), B11B65H09         (SEQ ID NOs:39, 41) and BII665603 (SEQ ID NOs:43, 45), or CDRs         which are identical or which have no more than two alterations         from said CDRs.         Herceptin (Genentech)—U.S. Pat. No. 6,054,297; ATCC accession         no. CRL-10463 (Genentech)

Pertuzumab (Genentech)

-   -   US20110117097         -   for example, see SEQ IDs No. 15&16, SEQ IDs No. 17&18, SEQ             IDs No. 23&24 & ATCC accession numbers HB-12215, HB-12216,             CRL 10463, HB-12697.     -   US20090285837     -   US20090202546         -   for example, ATCC accession numbers: HB-12215, HB-12216, CRL             10463, HB-12698.     -   US20060088523         -   for example, ATCC accession numbers: HB-12215, HB-12216         -   for example, an antibody comprising the variable light and             variable heavy amino acid sequences in SEQ ID Nos. 3 and 4,             respectively.         -   for example, an antibody comprising a light chain amino acid             sequence selected from SEQ ID No. 15 and 23, and a heavy             chain amino acid sequence selected from SEQ ID No. 16 and 24     -   US20060018899         -   for example, ATCC accession numbers: (7C2) HB-12215, (7F3)             HB-12216, (4D5) CRL-10463, (2C4) HB-12697.         -   for example, an antibody comprising the amino acid sequence             in SEQ ID No. 23, or a deamidated and/or oxidized variant             thereof.     -   US2011/0159014         -   for example, an antibody having a light chain variable             domain comprising the hypervariable regions of SEQ ID NO:             1″.         -   For example, an antibody having a heavy chain variable             domain comprising the hypervariable regions of SEQ ID NO: 2.     -   US20090187007         Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline     -   For example, see International Joint Cancer Institute and         Changhai Hospital Cancer Cent: HMTI-Fc Ab—Gao J., et al BMB Rep.         2009 Oct. 31; 42(10):636-41.

Symphogen: US20110217305 Union Stem Cell & Gene Engineering, China—Liu H Q., et al Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 May; 26(5):456-8. (18) NCA (CEACAM6) Nucleotide

Genbank accession no M18728 Genbank version no. M18728.1 GI:189084 Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59907 Genbank version no. AAA59907.1 GI:189085 Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004/063709; EP1439393 (Claim 7); WO2004/044178 (Example 4); WO2004/031238; WO2003/042661 (Claim 12); WO2002/78524 (Example 2); WO2002/86443 (Claim 27; Page 427); WO2002/60317 (Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728.

(19) MDP (DPEP1) Nucleotide

Genbank accession no BC017023 Genbank version no. BC017023.1 GI:16877538 Genbank record update date: Mar. 6, 2012 01:00 PM

Polypeptide

Genbank accession no. AAH17023 Genbank version no. AAH17023.1 GI:16877539 Genbank record update date: Mar. 6, 2012 01:00 PM

CROSS REFERENCES

Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003/016475 (Claim 1); WO2002/64798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO99/46284 (FIG. 9); MIM:179780.

(20) IL20R-alpha (IL20Ra, ZCYTOR7) Nucleotide

Genbank accession no AF184971 Genbank version no. AF184971.1 GI:6013324 Genbank record update date: Mar. 10, 2010 10:00 PM

Polypeptide

Genbank accession no. AAF01320 Genbank version no. AAF01320.1 GI:6013325 Genbank record update date: Mar. 10, 2010 10:00 PM

CROSS REFERENCES

Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003) Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004/005320 (Example 5); WO2003/029262 (Page 74-75); WO2003/002717 (Claim 2; Page 63); WO2002/22153 (Page 45-47); US2002/042366 (Page 20-21); WO2001/46261 (Page 57-59); WO2001/46232 (Page 63-65); WO98/37193 (Claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB) Nucleotide

Genbank accession no AF229053 Genbank version no. AF229053.1 GI:10798902 Genbank record update date: Mar. 11, 2010 12:58 AM

Polypeptide

Genbank accession no. AAG23135 Genbank version no. AAG23135.1 GI:10798903 Genbank record update date: Mar. 11, 2010 12:58 AM

CROSS REFERENCES

Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; US2003/186372 (Claim 11); US2003/186373 (Claim 11); US2003/119131 (Claim 1; FIG. 52); US2003/119122 (Claim 1; FIG. 52); US2003/119126 (Claim 1); US2003/119121 (Claim 1; FIG. 52); US2003/119129 (Claim 1); US2003/119130 (Claim 1); US2003/119128 (Claim 1; FIG. 52); US2003/119125 (Claim 1); WO2003/016475 (Claim 1); WO2002/02634 (Claim 1)

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) Nucleotide

Genbank accession no NM_004442 Genbank version no. NM_004442.6 GI:111118979 Genbank record update date: Sep. 8, 2012 04:43 PM

Polypeptide

Genbank accession no. NP_004433 Genbank version no. NP_004433.2 GI:21396504 Genbank record update date: Sep. 8, 2012 04:43 PM

CROSS REFERENCES

Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42); MIM:600997.

(23) ASLG659 (B7h) Nucleotide

Genbank accession no. AX092328 Genbank version no. AX092328.1 GI:13444478 Genbank record update date: Jan. 26, 2011 07:37 AM

CROSS REFERENCES

US2004/0101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003/165504 (Claim 1); US2003/124140 (Example 2); US2003/065143 (FIG. 60); WO2002/102235 (Claim 13; Page 299); US2003/091580 (Example 2); WO2002/10187 (Claim 6; FIG. 10); WO2001/94641 (Claim 12; FIG. 7b); WO2002/02624 (Claim 13; FIG. 1A-1B); US2002/034749 (Claim 54; Page 45-46); WO2002/06317 (Example 2; Page 320-321, Claim 34; Page 321-322); WO2002/71928 (Page 468-469); WO2002/02587 (Example 1; FIG. 1); WO2001/40269 (Example 3; Pages 190-192); WO2000/36107 (Example 2; Page 205-207); WO2004/053079 (Claim 12); WO2003/004989 (Claim 1); WO2002/71928 (Page 233-234, 452-453); WO 01/16318.

(24) PSCA (Prostate Stem Cell Antigen Precursor) Nucleotide

Genbank accession no AJ297436 Genbank version no. AJ297436.1 GI:9367211 Genbank record update date: Feb. 1, 2011 11:25 AM

Polypeptide

Genbank accession no. CAB97347 Genbank version no. CAB97347.1 GI:9367212 Genbank record update date: Feb. 1, 2011 11:25 AM

CROSS REFERENCES

Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004/022709; EP1394274 (Example 11); US2004/018553 (Claim 17); WO2003/008537 (Claim 1); WO2002/81646 (Claim 1; Page 164); WO2003/003906 (Claim 10; Page 288); WO2001/40309 (Example 1; FIG. 17); US2001/055751 (Example 1; FIG. 1b); WO2000/32752 (Claim 18; FIG. 1); WO98/51805 (Claim 17; Page 97); WO98/51824 (Claim 10; Page 94); WO98/40403 (Claim 2; FIG. 1B); Accession: 043653; EMBL; AF043498; AAC39607.1

(25) GEDA Nucleotide

Genbank accession no AY260763 Genbank version no. AY260763.1 GI:30102448 Genbank record update date: Mar. 11, 2010 02:24 AM

Polypeptide

Genbank accession no. AAP14954 Genbank version no. AAP14954.1 GI:30102449 Genbank record update date: Mar. 11, 2010 02:24 AM

CROSS REFERENCES

AP14954 lipoma HMGIC fusion-partnerlike protein/pid=AAP14954.1-Homo sapiens (human); WO2003/054152 (Claim 20); WO2003/000842 (Claim 1); WO2003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45); GI:30102449;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3)

Nucleotide

Genbank accession no AF116456 Genbank version no. AF116456.1 GI:4585274 Genbank record update date: Mar. 10, 2010 09:44 PM

Polypeptide

Genbank accession no. AAD25356 Genbank version no. AAD25356.1 GI:4585275 Genbank record update date: Mar. 10, 2010 09:44 PM

CROSS REFERENCES

BAFF receptor/pid=NP_443177.1-Homo sapiens: Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001); WO2004/058309; WO2004/011611; WO2003/045422 (Example; Page 32-33); WO2003/014294 (Claim 35; FIG. 6B); WO2003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col 136-137); WO2002/38766 (Claim 3; Page 133); WO2002/24909 (Example 3; FIG. 3); MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B-Cell Receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814)

Nucleotide

Genbank accession no AK026467 Genbank version no. AK026467.1 GI:10439337 Genbank record update date: Sep. 11, 2006 11:24 PM

Polypeptide

Genbank accession no. BAB15489 Genbank version no. BAB15489.1 GI:10439338 Genbank record update date: Sep. 11, 2006 11:24 PM

CROSS REFERENCES

Wilson et al (1991) J. Exp. Med. 173:137-146; WO2003/072036 (Claim 1; FIG. 1); IM:107266; NP_001762.1; NM0017711.

(27a) Cd22 (Cd22 Molecule) Nucleotide

Genbank accession no X52785 Genbank version no. X52785.1 GI:29778 Genbank record update date: Feb. 2, 2011 10:09 AM

Polypeptide

Genbank accession no. CAA36988 Genbank version no. CAA36988.1 GI:29779 Genbank record update date: Feb. 2, 2011 10:09 AM

CROSS REFERENCES

Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)

Other Information Official Symbol: CD22 Other Aliases: SIGLEC-2, SIGLEC2

Other Designations: B-cell receptor CD22; B-lymphocyte cell adhesion molecule; BL-CAM; CD22 antigen; T-cell surface antigen Leu-14; sialic acid binding Ig-like lectin 2; sialic acid-binding Ig-like lectin 2

Antibodies G5/44 (Inotuzumab): DiJoseph J F., et al Cancer Immunol Immunother. 2005 January; 54(1):11-24. Epratuzumab—Goldenberg D M., et al Expert Rev Anticancer Ther. 6(10): 1341-53, 2006.

(28) CD79a (CD79A, CD79alpha), Immunoglobulin-Associated Alpha, a B Cell-Specific Protein that Covalently Interacts with Ig Beta (CD79B) and Forms a Complex on the Surface with Ig M Molecules, Transduces a Signal Involved in B-Cell Differentiation), Pl: 4.84, MW: 25028 TM: 2

[P] Gene Chromosome: 19q13.2). Nucleotide

Genbank accession no NM_001783 Genbank version no. NM_001783.3 GI:90193587 Genbank record update date: Jun. 26, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001774 Genbank version no. NP_001774.1 GI:4502685 Genbank record update date: Jun. 26, 2012 01:48 PM

CROSS REFERENCES

WO2003/088808, US2003/0228319; WO2003/062401 (Claim 9); US2002/150573 (Claim 4, pages 13-14); WO99/58658 (Claim 13, FIG. 16); WO92/07574 (FIG. 1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531; Müller et al (1992) Eur. J. Immunol. 22:1621-1625; Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G Protein-Coupled Receptor that is Activated by the CXCL13 Chemokine, Functions in Lymphocyte Migration and Humoral Defense, Plays a Role in HIV-2 Infection and Perhaps Development of AIDS, Lymphoma, Myeloma, and Leukemia); 372 Aa, Pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,

Nucleotide

Genbank accession no NM_001716 Genbank version no. NM_001716.4 GI:342307092 Genbank record update date: Sep. 30, 2012 01:49 PM

Polypeptide

Genbank accession no. NP_001707 Genbank version no. NP_001707.1 GI:4502415 Genbank record update date: Sep. 30, 2012 01:49 PM

CROSS REFERENCES

WO2004/040000; WO2004/015426; US2003/105292 (Example 2); U.S. Pat. No. 6,555,339 (Example 2); WO2002/61087 (FIG. 1); WO2001/57188 (Claim 20, page 269); WO2001/72830 (pages 12-13); WO2000/22129 (Example 1, pages 152-153, Example 2, pages 254-256); WO99/28468 (Claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO94/28931 (pages 56-58); WO92/17497 (Claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol. 22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779

(30) HLA-DOB (Beta Subunit of MHC Class II Molecule (Ia Antigen) that Binds Peptides and Presents them to CD4+T Lymphocytes); 273 Aa, Pl: 6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3)

Nucleotide

Genbank accession no NM_002120 Genbank version no. NM_002120.3 GI:118402587 Genbank record update date: Sep. 8, 2012 04:46 PM

Polypeptide

Genbank accession no. NP_002111 Genbank version no. NP_002111.1 GI:4504403 Genbank record update date: Sep. 8, 2012 04:46 PM

CROSS REFERENCES

Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989) Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol. 228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766; Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) Tissue Antigens 59:512-519; WO99/58658 (Claim 13, FIG. 15); U.S. Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics 30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

(31) P2X5 (Purinergic Receptor P2X Ligand-Gated Ion Channel 5, an Ion Channel Gated by Extracellular ATP, May be Involved in Synaptic Transmission and Neurogenesis, Deficiency May Contribute to the Pathophysiology of Idiopathic Detrusor Instability); 422 Aa), Pl: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).

Nucleotide

Genbank accession no NM_002561 Genbank version no. NM_002561.3 GI:325197202 Genbank record update date: Jun. 27, 2012 12:41 AM

Polypeptide

Genbank accession no. NP_002552 Genbank version no. NP_002552.2 GI:28416933 Genbank record update date: Jun. 27, 2012 12:41 AM

CROSS REFERENCES

Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004/047749; WO2003/072035 (Claim 10); Touchman et al (2000) Genome Res. 10:165-173; WO2002/22660 (Claim 20); WO2003/093444 (Claim 1); WO2003/087768 (Claim 1); WO2003/029277 (page 82)

(32) CD72 (B-Cell Differentiation Antigen CD72, Lyb-2); 359 Aa, Pl: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3). Nucleotide

Genbank accession no NM_001782 Genbank version no. NM_001782.2 GI:194018444 Genbank record update date: Jun. 26, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_001773 Genbank version no. NP_001773.1 GI:4502683 Genbank record update date: Jun. 26, 2012 01:43 PM

CROSS REFERENCES

WO2004042346 (Claim 65); WO2003/026493 (pages 51-52, 57-58); WO2000/75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903.

(33) LY64 (Lymphocyte Antigen 64 (RP105), Type I Membrane Protein of the Leucine Rich Repeat (LRR) Family, Regulates B-Cell Activation and Apoptosis, Loss of Function is Associated with Increased Disease Activity in Patients with Systemic Lupus Erythematosis); 661 Aa, Pl: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).

Nucleotide

Genbank accession no NM_005582 Genbank version no. NM_005582.2 GI:167555126 Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_005573 Genbank version no. NP_005573.2 GI:167555127 Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS REFERENCES

US2002/193567; WO97/07198 (Claim 11, pages 39-42); Miura et al (1996) Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822; WO2003/083047; WO97/44452 (Claim 8, pages 57-61); WO2000/12130 (pages 24-26).

(34) FcRH1 (Fc Receptor-Like Protein 1, a Putative Receptor for the Immunoglobulin Fc Domain that Contains C2 Type Ig-Like and ITAM Domains, May have a Role in B-Lymphocyte Differentiation); 429 Aa, Pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)

Nucleotide

Genbank accession no NM_052938 Genbank version no. NM_052938.4 GI:226958543 Genbank record update date: Sep. 2, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_443170 Genbank version no. NP_443170.1 GI:16418419 Genbank record update date: Sep. 2, 2012 01:43 PM

CROSS REFERENCES

WO2003/077836; WO2001/38490 (Claim 6, FIG. 18E-1-18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003/089624 (Claim 8); EP1347046 (Claim 1); WO2003/089624 (Claim 7).

(35) IRTA2 (Immunoglobulin Superfamily Receptor Translocation Associated 2, a Putative Immunoreceptor with Possible Roles in B Cell Development and Lymphoma Genesis; Deregulation of the Gene by Translocation Occurs in Some B Cell Malignancies); 977 Aa, Pl: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21)

Nucleotide

Genbank accession no AF343662 Genbank version no. AF343662.1 GI:13591709 Genbank record update date: Mar. 11, 2010 01:16 AM

Polypeptide

Genbank accession no. AAK31325 Genbank version no. AAK31325.1 GI:13591710 Genbank record update date: Mar. 11, 2010 01:16 AM

CROSS REFERENCES

AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL⁸³⁴¹⁸⁷, AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571.1; WO2003/024392 (Claim 2, FIG. 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003/077836; WO2001/38490 (Claim 3, FIG. 18B-1-18B-2).

(36) TENB2 (TMEFF2, Tomoregulin, TPEF, HPP1, TR, Putative Transmembrane Proteoglycan, Related to the EGF/Heregulin Family of Growth Factors and Follistatin); 374 Aa) Nucleotide

Genbank accession no AF179274 Genbank version no. AF179274.2 GI:12280939 Genbank record update date: Mar. 11, 2010 01:05 AM

Polypeptide

Genbank accession no. AAD55776 Genbank version no. AAD55776.2 GI:12280940 Genbank record update date: Mar. 11, 2010 01:05 AM

CROSS REFERENCES

NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; AY358907, CAF85723, CQ782436; WO2004/074320; JP2004113151; WO2003/042661; WO2003/009814; EP1295944 (pages 69-70); WO2002/30268 (page 329); WO2001/90304; US2004/249130; US2004/022727; WO2004/063355; US2004/197325; US2003/232350; US2004/005563; US2003/124579; Horie et al (2000) Genomics 67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J Cancer. October 15; 94(2):178-84.

(37) PSMA—FOLH1 (Folate Hydrolase (Prostate-Specific Membrane Antigen) 1) Nucleotide

Genbank accession no M99487 Genbank version no. M99487.1 GI:190663 Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA60209 Genbank version no. AAA60209.1 GI:190664 Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Israeli R. S., et al Cancer Res. 53 (2), 227-230 (1993)

Other Information Official Symbol: FOLH1

Other Aliases: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM, PSMA, mGCP Other Designations: N-acetylated alpha-linked acidic dipeptidase 1; N-acetylated-alpha-linked acidic dipeptidase I; NAALADase I; cell growth-inhibiting gene 27 protein; folylpoly-gamma-glutamate carboxypeptidase; glutamate carboxylase II; glutamate carboxypeptidase 2; glutamate carboxypeptidase II; membrane glutamate carboxypeptidase; prostate specific membrane antigen variant F; pteroylpoly-gamma-glutamate carboxypeptidase

Antibodies

U.S. Pat. No. 7,666,425: Antibodies produces by Hybridomas having the following ATCC references:ATCC accession No. HB-12101, ATCC accession No. HB-12109, ATCC accession No. HB-12127 and ATCC accession No. HB-12126. Proscan: a monoclonal antibody selected from the group consisting of 8H12, 3E11, 17G1, 2964, 30C1 and 20F2 (U.S. Pat. No. 7,811,564; Moffett S., et al Hybridoma (Larchmt). 2007 December; 26(6):363-72). Cytogen: monoclonal antibodies 7E11-05 (ATCC accession No. HB 10494) and 9H10-A4 (ATCC accession No. HB11430)—U.S. Pat. No. 5,763,202 GlycoMimetics: NUH2-ATCC accession No. HB 9762 (U.S. Pat. No. 7,135,301) Human Genome Science: HPRAJ70-ATCC accession No. 97131 (U.S. Pat. No. 6,824,993); Amino acid sequence encoded by the cDNA clone (HPRAJ70) deposited as American Type Culture Collection (“ATCC”) Deposit No. 97131 Medarex: Anti-PSMA antibodies that lack fucosyl residues—U.S. Pat. No. 7,875,278

Mouse anti-PSMA antibodies include the 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9, and monoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6 or 4C8B9 have been publicly deposited and are described in U.S. Pat. No. 6,159,508. Relevant hybridomas have been publicly deposited and are described in U.S. Pat. No. 6,107,090. Moreover, humanized anti-PSMA antibodies, including a humanized version of J591, are described in further detail in PCT Publication WO 02/098897.

Other mouse anti-human PSMA antibodies have been described in the art, such as mAb 107-1A4 (Wang, S. et al. (2001) Int. J. Cancer 92:871-876) and mAb 2C9 (Kato, K. et al. (2003) Int. J. Urol. 10:439-444).

Examples of human anti-PSMA monoclonal antibodies include the 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 antibodies, isolated and structurally characterized as originally described in PCT Publications WO 01/09192 and WO 03/064606 and in U.S. Provisional Application Ser. No. 60/654,125, entitled “Human Monoclonal Antibodies to Prostate Specific Membrane Antigen (PSMA)”, filed on Feb. 18, 2005. The V.sub.H amino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 1-9, respectively. The V.sub.L amino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include the antibodies disclosed in PCT Publication WO 03/034903 and US Application No. 2004/0033229.

NW Biotherapeutics: A hybridoma cell line selected from the group consisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.l. having ATCC accession number HB12309, 4E10-1.14 having ATCC accession number HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487), 3E6 (ATCC HB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3 (ATCC HB12489), 3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC HB12493), 1G9 (ATCC HB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485)—see U.S. Pat. No. 6,150,508

PSMA Development Company/Progenics/Cytogen—Seattle Genetics: mAb 3.9, produced by the hybridoma deposited under ATCC Accession No. PTA-3258 or mAb 10.3, produced by the hybridoma deposited under ATCC Accession No. PTA-3347-U.S. Pat. No. 7,850,971

PSMA Development Company—Compositions of PSMA antibodies (US 20080286284, Table 1)

-   -   This application is a divisional of U.S. patent application Ser.         No. 10/395,894, filed on Mar. 21, 2003 (U.S. Pat. No. 7,850,971)

University Hospital Freiburg, Germany—mAbs 3/A12, 3/E7, and 3/F11 (Wolf P., et al Prostate. 2010 April 1; 70(5):562-9).

(38) SST (Somatostatin Receptor; Note that there are 5 Subtypes)

(38.1) SSTR2 (Somatostatin Receptor 2) Nucleotide

Genbank accession no NM_001050 Genbank version no. NM_001050.2 GI:44890054 Genbank record update date: Aug. 19, 2012 01:37 PM

Polypeptide

Genbank accession no. NP_001041 Genbank version no. NP_001041.1 GI:4557859 Genbank record update date: Aug. 19, 2012 01:37 PM

CROSS REFERENCES

Yamada Y., et al Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255 (1992); Susini C., et al Ann Oncol. 2006 December; 17(12):1733-42

Other Information Official Symbol: SSTR2

Other Designations: SRIF-1; SS2R; somatostatin receptor type 2 (38.2) SSTR5 (Somatostatin receptor 5)

Nucleotide

Genbank accession no D16827 Genbank version no. D16827.1 GI:487683 Genbank record update date: Aug. 1, 2006 12:45 PM

Polypeptide

Genbank accession no. BAA04107 Genbank version no. BAA04107.1 GI:487684 Genbank record update date: Aug. 1, 2006 12:45 PM

CROSS REFERENCES

Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852 (1993)

Other Information Official Symbol: SSTR5 Other Aliases: SS-5-R

Other Designations: Somatostatin receptor subtype 5; somatostatin receptor type 5

(38.3) Sstr1 (38.4)SSTR3 (38.5) SSTR4 AvB6—Both Subunits (39+40) (39) ITGAV (Integrin, Alpha V; Nucleotide

Genbank accession no M14648 J02826 M18365 Genbank version no. M14648.1 GI:340306 Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA36808 Genbank version no. AAA36808.1 GI:340307 Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS REFERENCES

Suzuki S., et al Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618 (1986)

Other Information Official Symbol: ITGAV Other Aliases: CD51, MSK8, VNRA, VTNR

Other Designations: antigen identified by monoclonal antibody L230; integrin alpha-V; integrin alphaVbeta3; integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51); vitronectin receptor subunit alpha

(40) ITGB6 (Integrin, Beta 6) Nucleotide

Genbank accession no NM_000888 Genbank version no. NM_000888.3 GI:9966771 Genbank record update date: Jun. 27, 2012 12:46 AM

Polypeptide

Genbank accession no. NP_000879 Genbank version no. NP_000879.2 GI:9625002 Genbank record update date: Jun. 27, 2012 12:46 AM

CROSS REFERENCES

Sheppard D. J., et al Biol. Chem. 265 (20), 11502-11507 (1990)

Other Information Official Symbol: ITGB6

Other Designations: integrin beta-6

Antibodies

Biogen: U.S. Pat. No. 7,943,742-Hybridoma clones 6.3G9 and 6.8G6 were deposited with the ATCC, accession numbers ATCC PTA-3649 and -3645, respectively. Biogen: U.S. Pat. No. 7,465,449-In some embodiments, the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.261, 6.2610, 6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.105. Centocor (J&J): U.S. Pat. No. 7,550,142; U.S. Pat. No. 7,163,681

-   -   For example in U.S. Pat. No. 7,550,142-an antibody having human         heavy chain and human light chain variable regions comprising         the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Seattle Genetics: 15H3 (Ryan M C., et al Cancer Res Apr. 15, 2012; 72(8 Supplement): 4630) (41) CEACAM5 (Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5) Nucleotide

Genbank accession no M17303 Genbank version no. M17303.1 GI:178676 Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAB59513 Genbank version no. AAB59513.1 GI:178677 Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987)

Other Information Official Symbol: CEACAM5 Other Aliases: CD66e, CEA

Other Designations: meconium antigen 100

Antibodies AstraZeneca-MedImmune:US 20100330103; US20080057063;

-   -   US20020142359         -   for example an antibody having complementarity determining             regions (CDRs) with the following sequences: heavy chain;             CDR1-DNYMH, CDR2-WIDPENGDTE YAPKFRG, CDR3-LIYAGYLAMD Y; and             light chain CDR1-SASSSVTYMH, CDR2-STSNLAS, CDR3—QQRSTYPLT.     -   Hybridoma 806.077 deposited as European Collection of Cell         Cultures (ECACC) deposit no. 96022936.         Research Corporation Technologies, Inc.: U.S. Pat. No. 5,047,507         Bayer Corporation: U.S. Pat. No. 6,013,772         BioAlliance: U.S. Pat. No. 7,982,017; U.S. Pat. No. 7,674,605     -   U.S. Pat. No. 7,674,605         -   an antibody comprising the heavy chain variable region             sequence from the amino acid sequence of SEQ ID NO: 1, and             the light chain variable region sequence from the amino acid             sequence of SEQ ID NO:2.         -   an antibody comprising the heavy chain variable region             sequence from the amino acid sequence of SEQ ID NO:5, and             the light chain variable region sequence from the amino acid             sequence of SEQ ID NO:6.             Celltech Therapeutics Limited: U.S. Pat. No. 5,877,293             The Dow Chemical Company: U.S. Pat. No. 5,472,693; U.S. Pat.             No. 6,417,337; U.S. Pat. No. 6,333,405     -   U.S. Pat. No. 5,472,693—for example, ATCC No. CRL-11215     -   U.S. Pat. No. 6,417,337—for example, ATCC CRL-12208     -   U.S. Pat. No. 6,333,405—for example, ATCC CRL-12208         Immunomedics, Inc: U.S. Pat. No. 7,534,431; U.S. Pat. No.         7,230,084; U.S. Pat. No. 7,300,644; U.S. Pat. No. 6,730,300;     -   US20110189085         -   an antibody having CDRs of the light chain variable region             comprise: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20); CDR2             comprises WTSTRHT (SEQ ID NO: 21); and CDR3 comprises             QQYSLYRS (SEQ ID NO: 22);         -   and the CDRs of the heavy chain variable region of said             anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID NO:             23); CDR2 comprises EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and             CDR3 comprises LYFGFPWFAY (SEQ ID NO: 25).     -   US20100221175; US20090092598; US20070202044; US20110064653;         US20090185974; US20080069775.

(42) MET (Met Proto-Oncogene; Hepatocyte Growth Factor Receptor) Nucleotide

Genbank accession no M35073 Genbank version no. M35073.1 GI:187553 Genbank record update date: Mar. 6, 2012 11:12 AM

Polypeptide

Genbank accession no. AAA59589 Genbank version no. AAA59589.1 GI:553531 Genbank record update date: Mar. 6, 2012 11:12 AM

CROSS REFERENCES

Dean M., et al Nature 318 (6044), 385-388 (1985)

Other Information Official Symbol: MET

Other Aliases: AUTS9, HGFR, RCCP2, c-Met Other Designations: HGF receptor; HGF/SF receptor; SF receptor; hepatocyte growth factor receptor; met proto-oncogene tyrosine kinase; proto-oncogene c-Met; scatter factor receptor; tyrosine-protein kinase Met

Antibodies Abgenix/Pfizer: US20100040629

-   -   for example, the antibody produced by hybridoma 13.3.2 having         American Type Culture Collection (ATCC) accession number         PTA-5026; the antibody produced by hybridoma 9.1.2 having ATCC         accession number PTA-5027; the antibody produced by hybridoma         8.70.2 having ATCC accession number PTA-5028; or the antibody         produced by hybridoma 6.90.3 having ATCC accession number         PTA-5029.

Amgen/Pfizer: US20050054019

-   -   for example, an antibody comprising a heavy chain having the         amino acid sequences set forth in SEQ ID NO: 2 where X2 is         glutamate and X4 is serine and a light chain having the amino         acid sequence set forth in SEQ ID NO: 4 where X8 is alanine,         without the signal sequences; an antibody comprising a heavy         chain having the amino acid sequences set forth in SEQ ID NO: 6         and a light chain having the amino acid sequence set forth in         SEQ ID NO: 8, without the signal sequences; an antibody         comprising a heavy chain having the amino acid sequences set         forth in SEQ ID NO: 10 and a light chain having the amino acid         sequence set forth in SEQ ID NO: 12, without the signal         sequences; or an antibody comprising a heavy chain having the         amino acid sequences set forth in SEQ ID NO: 14 and a light         chain having the amino acid sequence set forth in SEQ ID NO: 16,         without the signal sequences.

Agouron Pharmaceuticals (Now Pfizer): US20060035907 Eli Lilly: US20100129369

Genentech: U.S. Pat. No. 5,686,292; US20100028337; US20100016241; US20070129301; US20070098707; US20070092520, US20060270594; US20060134104; US20060035278; US20050233960; US20050037431

-   -   U.S. Pat. No. 5,686,292—for example, ATCC HB-11894 and ATCC         HB-11895 US 20100016241—for example, ATCC HB-11894 (hybridoma         1A3.3.13) or HB-11895 (hybridoma 5D5.11.6)

National Defense Medical Center, Taiwan: Lu R M., et al Biomaterials. 2011 Apr.; 32(12):3265-74. Novartis: US20090175860

-   -   for example, an antibody comprising the sequences of CDR1, CDR2         and CDR3 of heavy chain 4687, wherein the sequences of CDR1,         CDR2, and CDR3 of heavy chain 4687 are residues 26-35, 50-65,         and 98-102, respectively, of SEQ ID NO: 58; and the sequences of         CDR1, CDR2, and CDR3 of light chain 5097, wherein the sequences         of CDR1, CDR2, and CDR3 oflight chain 5097 are residues 24-39,         55-61, and 94-100 of SEQ ID NO: 37.

Pharmacia Corporation: US20040166544 Pierre Fabre: US20110239316, US20110097262, US20100115639

Sumsung: US 20110129481—for example a monoclonal antibody produced from a hybridoma cell having accession number KCLRF-BP-00219 or accession number of KCLRF-BP-00223. Samsung: US 20110104176—for example an antibody produced by a hybridoma cell having Accession Number: KCLRF-BP-00220.

University of Turin Medical School: DN-30 Pacchiana G., et al J Biol Chem. 2010 Nov. 12; 285(46):36149-57 Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005 September; 31(1):41-54. (43) MUC1 (Mucin 1, Cell Surface Associated) Nucleotide

Genbank accession no J05581 Genbank version no. J05581.1 GI:188869 Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59876 Genbank version no. AAA59876.1 GI:188870 Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Gendler S. J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990)

Other Information Official Symbol: MUC1 Other Aliases: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1, MUC-1/SEC, MUC-1/X, MUC1/ZD, PEM, PEMT, PUM

Other Designations: DF3 antigen; H23 antigen; breast carcinoma-associated antigen DF3; carcinoma-associated mucin; episialin; krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; peanut-reactive urinary mucin; polymorphic epithelial mucin; tumor associated epithelial mucin; tumor-associated epithelial membrane antigen; tumor-associated mucin

Antibodies

AltaRex—Quest Pharma Tech: U.S. Pat. No. 6,716,966—for example an Alt-1 antibody produced by the hybridoma ATCC No PTA-975. AltaRex—Quest Pharma Tech: U.S. Pat. No. 7,147,850

CRT: 5E5-Sørensen AL., et al Glycobiology vol. 16 no. 2 pp. 96-107, 2006; HMFG2—Burchell J., et al Cancer Res., 47, 5476-5482 (1987)

Glycotope GT-MAB: GT-MAB 2.5-GEX (Website: http://www.glycotope.com/pipeline/pankomab-gex) Immunogen: U.S. Pat. No. 7,202,346

-   -   for example, antibody MJ-170: hybridoma cell line MJ-170 ATCC         accession no. PTA-5286Monoclonal antibody MJ-171: hybridoma cell         line MJ-171 ATCC accession no. PTA-5287; monoclonal antibody         MJ-172: hybridoma cell line MJ-172 ATCC accession no. PTA-5288;         or monoclonal antibody MJ-173: hybridoma cell line MJ-173 ATCC         accession no. PTA-5302         Immunomedics: U.S. Pat. No. 6,653,104         Ramot Tel Aviv Uni: U.S. Pat. No. 7,897,351         Regents Uni. CA: U.S. Pat. No. 7,183,388; US20040005647;         US20030077676.         Roche GlycArt: U.S. Pat. No. 8,021,856

Russian National Cancer Research Center: Imuteran—Ivanov P K., et al Biotechnol J. 2007 July; 2(7):863-70

Technische Univ Braunschweig: (11136, HT186-B7, HT186-D11, HT186-G2, HT200-3A-C1, HT220-M-D1, HT220-M-G8)—Thie H., et al PLoS One. 2011 Jan. 14; 6(1):e15921

(44) CA9 (Carbonic Anhydrase IX) Nucleotide

Genbank accession no. X66839 Genbank version no. X66839.1 GI:1000701 Genbank record update date: Feb. 2, 2011 10:15 AM

Polypeptide

Genbank accession no. CAA47315 Genbank version no. CAA47315.1 GI:1000702 Genbank record update date: Feb. 2, 2011 10:15 AM

CROSS REFERENCES

Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994)

Other Information Official Symbol: CA9 Other Aliases: CAIX, MN

Other Designations: CA-IX; P54/58N; RCC-associated antigen G250; RCC-associated protein G250; carbonate dehydratase IX; carbonic anhydrase 9; carbonic dehydratase; membrane antigen MN; pMW1; renal cell carcinoma-associated antigen G250

Antibodies Abgenix/Amgen: US20040018198

Affibody: Anti-CAIX Affibody molecules

-   -   (http://www.affibody.com/en/Product-Portfolio/Pipeline/)         Bayer: U.S. Pat. No. 7,462,696         Bayer/Morphosys: 3ee9 mAb—Petrul H M., et al Mol Cancer Ther.         2012 February; 11(2):340-9         Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57,         G106, G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010         Mar. 10; 5(3):e9625         Institute of Virology, Slovak Academy of Sciences (Bayer)—U.S.         Pat. No. 5,955,075     -   for example, M75-ATCC Accession No. HB 11128 or MN₁₂—ATCC         Accession No. HB 11647         Institute of Virology, Slovak Academy of Sciences: U.S. Pat. No.         7,816,493     -   for example the M75 monoclonal antibody that is secreted from         the hybridoma VU-M75, which was deposited at the American Type         Culture Collection under ATCC No. HB 11128; or the V/10         monoclonal antibody secreted from the hybridoma V/10-VU, which         was deposited at the International Depository Authority of the         Belgian Coordinated Collection of Microorganisms (BCCM) at the         Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP)         at the Universeit Gent in Gent, Belgium, under Accession No.         LMBP 6009CB.

Institute of Virology, Slovak Academy of Sciences US20080177046; US20080176310; US20080176258; US20050031623 Novartis: US20090252738

Wilex: U.S. Pat. No. 7,691,375—for example the antibody produced by the hybridoma cell line DSM ASC 2526.

Wilex: US20110123537; Rencarex: Kennett R H., et al Curr Opin Mol Ther. 2003 February; 5(1):70-5 Xencor: US20090162382 (45) EGFRvIII (Epidermal Growth Factor Receptor (EGFR), Transcript Variant 3, Nucleotide

Genbank accession no. NM_201283 Genbank version no. NM_201283.1 GI:41327733 Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_958440 Genbank version no. NP_958440.1 GI:41327734 Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

Batra S K., et al Cell Growth Differ 1995; 6:1251-1259.

Antibodies:

U.S. Pat. No. 7,628,986 and U.S. Pat. No. 7,736,644 (Amgen)

-   -   For example, a heavy chain variable region amino acid sequence         selected from the group consisting of SEQ ID NO: 142 and         variants & a light chain variable region amino acid sequence         selected from the group consisting of: SEQ ID NO: 144 and         variants.

US20100111979 (Amgen)

-   -   For example, an antibody comprising a heavy chain amino acid         sequence comprising:     -   CDR1 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR1 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);         CDR2 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR2 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);         and     -   CDR3 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR3 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17).

US20090240038 (Amgen)

-   -   For example, an antibody having at least one of the heavy or         light chain polypeptides comprises an amino acid sequence that         is at least 90% identical to the amino acid sequence selected         from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ         ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US20090175887 (Amgen)

-   -   For example, an antibody having a heavy chain amino acid         sequence selected from the group consisting of the heavy chain         amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131         (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ         ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID         NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID         NO: 16), and 333 (SEQ ID NO: 17).

US20090156790 (Amgen)

-   -   For example, antibody having heavy chain polypeptide and a light         chain polypeptide, wherein at least one of the heavy or light         chain polypeptides comprises an amino acid sequence that is at         least 90% identical to the amino acid sequence selected from the         group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO:         142, SEQ ID NO: 144, and any combination thereof.

US20090155282, US20050059087 and US20050053608 (Amgen)

-   -   For example, an antibody heavy chain amino acid sequence         selected from the group consisting of the heavy chain amino acid         sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO:         2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7),         250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12),         124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16),         and 333 (SEQ ID NO: 17).         MR1-1 (U.S. Pat. No. 7,129,332; Duke)     -   For example, a variant antibody having the sequence of SEQ ID         NO. 18 with the substitutions S₉₈P-T99Y in the CDR3 VH, and F92W         in CDR3 VL.

L⁸A4, H10, Y10 (Wikstrand C J., et al Cancer Res. 1995 Jul. 15; 55(14):3140-8; Duke) US20090311803 (Harvard University)

-   -   For example, SEQ ID NO:9 for antibody heavy chain variable         region, and SEQ ID NO: 3 for light chain variable region amino         acid sequences         US20070274991 (EMD72000, also known as matuzumab; Harvard         University)     -   For example, SEQ ID NOs: 3 & 9 for light chain and heavy chain         respectively         U.S. Pat. No. 6,129,915 (Schering)     -   For example, SEQ. ID NOs: 1, 2, 3, 4, 5 and 6.

mAb CH12-Wang H., et al FASEB J. 2012 January; 26(1):73-80 (Shanghai Cancer Institute). RAbDMvIII—Gupta P., et al BMC Biotechnol. 2010 Oct. 7; 10:72 (Stanford University Medical Center). mAb Ua30-Ohman L., et al Tumour Biol. 2002 March-April; 23(2):61-9 (Uppsala University). Han D G., et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 January; 30(1):25-9 (Xi'an Jiaotong University). (46) Cd33 (Cd33 Molecule) Nucleotide

Genbank accession no. M23197 Genbank version no. NM_23197.1 GI:180097 Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA51948 Genbank version no. AAA51948.1 GI:188098 Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

Simmons D., et al J. Immunol. 141 (8), 2797-2800 (1988)

Other Information Official Symbol: CD33 Other Aliases: SIGLEC-3, SIGLEC3, p67

Other Designations: CD33 antigen (gp67); gp67; myeloid cell surface antigen CD33; sialic acid binding Ig-like lectin 3; sialic acid-binding Ig-like lectin

Antibodies

H195 (Lintuzumab)—Raza A., et al Leuk Lymphoma. 2009 August; 50(8):1336-44; U.S. Pat. No. 6,759,045 (Seattle Genetics/Immunomedics)

mAb OKT9: Sutherland, D. R. et al. Proc Natl Acad Sci USA 78(7): 4515-4519 1981, Schneider, C., et al J Biol Chem 257, 8516-8522 (1982)

mAb E6: Hoogenboom, H. R., et al J Immunol 144, 3211-3217 (1990)

U.S. Pat. No. 6,590,088 (Human Genome Sciences)

-   -   For example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521         U.S. Pat. No. 7,557,189 (Immunogen)     -   For example, an antibody or fragment thereof comprising a heavy         chain variable region which comprises three CDRs having the         amino acid sequences of SEQ ID NOs:1-3 and a light chain         variable region comprising three CDRs having the amino acid         sequences of SEQ ID NOs:4-6.

(47) Cd19 (Cd19 Molecule) Nucleotide

Genbank accession no. NM_001178098 Genbank version no. NM_001178098.1 GI:296010920 Genbank record update date: Sep. 10, 2012 12:43 AM

Polypeptide

Genbank accession no. NP_001171569 Genbank version no. NP_001171569.1 GI:296010921 Genbank record update date: Sep. 10, 2012 12:43 AM

CROSS-REFERENCES

Tedder T F., et al J. Immunol. 143 (2): 712-7 (1989)

Other Information Official Symbol: CD19 Other Aliases: B4, CVID3

Other Designations: B-lymphocyte antigen CD19; B-lymphocyte surface antigen B4; T-cell surface antigen Leu-12; differentiation antigen CD19

Antibodies Immunogen: HuB4-Al-Katib A M., et al Clin Cancer Res. 2009 Jun. 15; 15(12):4038-45. 4G7: Kügler M., et al Protein Eng Des Sel. 2009 March; 22(3):135-47

-   -   For example, sequences in FIG. 3 of of Knappik, A. et al. J Mol         Biol 2000 February; 296(1):57-86

AstraZeneca/MedImmune: MEDI-551-Herbst R., et al J Pharmacol Exp Ther. 2010 October; 335(1):213-22 Glenmark Pharmaceuticals: GBR-401-Hou S., et al Mol Cancer Ther November 2011 10 (Meeting Abstract Supplement) C164

U.S. Pat. No. 7,109,304 (Immunomedics)

-   -   For example, an antibody comprising the sequence of hA19Vk (SEQ         ID NO:7) and the sequence of hA19VH (SEQ ID NO:10)         U.S. Pat. No. 7,902,338 (Immunomedics)     -   For example, an antibody or antigen-binding fragment thereof         that comprises the light chain complementarity determining         region CDR sequences CDR1 of SEQ ID NO: 16 (KASQSVDYDGDSYLN);         CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ ID NO: 18         (QQSTEDPWT) and the heavy chain CDR sequences CDR1 of SEQ ID NO:         19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3         of SEQ ID NO: 21 (RETTTVGRYYYAMDY) and also comprises human         antibody framework (FR) and constant region sequences with one         or more framework region amino acid residues substituted from         the corresponding framework region sequences of the parent         murine antibody, and wherein said substituted FR residues         comprise the substitution of serine for phenylalanine at Kabat         residue 91 of the heavy chain variable region.

Medarex: MDX-1342—Cardarelli P M., et al Cancer Immunol Immunother. 2010 February; 59(2):257-65. MorphoSys/Xencor: MOR-208/XmAb-5574-Zalevsky J., et al Blood. 2009 Apr. 16; 113(16):3735-43

U.S. Pat. No. 7,968,687 (Seattle Genetics)

-   -   An antibody or antigen-binding fragment comprising a heavy chain         variable domain comprising the amino acid sequence of SEQ ID         NO:9 and a light chain variable domain comprising the amino acid         sequence of SEQ ID NO: 24.         4G7 chim—Lang P., et al Blood. 2004 May 15; 103(10):3982-5         (University of Tübingen)

For example, FIG. 6 and SEQ ID No: 80 of US20120082664

Zhejiang University School of Medicine: 2E8-Zhang J., et al J Drug Target. 2010 November; 18(9):675-8 (48) IL2RA (Interleukin 2 Receptor, Alpha); NCBI Reference Sequence: NM_000417.2); Nucleotide

Genbank accession no. NM_000417 Genbank version no. NM_000417.2 GI:269973860 Genbank record update date: Sep. 9, 2012 04:59 PM

Polypeptide

Genbank accession no. NP_000408 Genbank version no. NP_000408.1 GI:4557667 Genbank record update date: Sep. 9, 2012 04:59 PM

CROSS-REFERENCES

Kuziel W. A., et al J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S (1990)

Other Information Official Symbol: IL2RA Other Aliases: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR

Other Designations: FIL-2 receptor subunit alpha; IL-2-RA; IL-2R subunit alpha; IL2-RA; TAC antigen; interleukin-2 receptor subunit alpha; p55

Antibodies

U.S. Pat. No. 6,383,487 (Novartis/UCL: Baxilisimab [Simulect]) U.S. Pat. No. 6,521,230 (Novartis/UCL: Baxilisimab [Simulect])

-   -   For example, an antibody having an antigen binding site         comprises at least one domain which comprises CDR1 having the         amino acid sequence in SEQ. ID. NO: 7, CDR2 having the amino         acid sequence in SEQ. ID. NO: 8, and CDR3 having the amino acid         sequence in SEQ. ID. NO: 9; or said CDR1, CDR2 and CDR3 taken in         sequence as a whole comprise an amino acid sequence which is at         least 90% identical to SEQ. ID. NOs: 7, 8 and 9 taken in         sequence as a whole.

Daclizumab—Rech A J., et al Ann N Y Acad Sci. 2009 September; 1174:99-106 (Roche) (49) AXL (AXL Receptor Tyrosine Kinase) Nucleotide

Genbank accession no. M76125 Genbank version no. M76125.1 GI:292869 Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA61243 Genbank version no. AAA61243.1 GI:29870 Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS-REFERENCES

O'Bryan J. P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991); Bergsagel P. L., et al J. Immunol. 148 (2), 590-596 (1992)

Other Information Official Symbol: AXL Other Aliases: JTK11, UFO

Other Designations: AXL oncogene; AXL transforming sequence/gene; oncogene AXL; tyrosine-protein kinase receptor UFO

Antibodies YW327.6S2-Ye X., et al Oncogene. 2010 Sep. 23; 29(38):5254-64. (Genentech)

BergenBio: BGB324 (http://www.bergenbio.com/BGB324)

(50) CD30-TNFRSF8 (Tumor Necrosis Factor Receptor Superfamily, Member 8) Nucleotide

Genbank accession no. M83554 Genbank version no. M83554.1 GI:180095 Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA51947 Genbank version no. AAA51947.1 GI:180096 Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS-REFERENCES

Durkop H., et al Cell 68 (3), 421-427 (1992)

Other Information Official Symbol: TNFRSF8 Other Aliases: CD30, D1S166E, Ki-1

Other Designations: CD30L receptor; Ki-1 antigen; cytokine receptor CD30; lymphocyte activation antigen CD30; tumor necrosis factor receptor superfamily member 8

(51) BCMA (B-Cell Maturation Antigen)—TNFRSF17 (Tumor Necrosis Factor Receptor Superfamily, Member 17) Nucleotide

Genbank accession no. Z29574 Genbank version no. Z29574.1 GI:471244 Genbank record update date: Feb. 2, 2011 10:40 AM

Polypeptide

Genbank accession no. CAA82690 Genbank version no. CAA82690.1 GI:471245 Genbank record update date: Feb. 2, 2011 10:40 AM

CROSS-REFERENCES

Laabi Y., et al Nucleic Acids Res. 22 (7), 1147-1154 (1994)

Other Information Official Symbol: TNFRSF17 Other Aliases: BCM, BCMA, CD269

Other Designations: B cell maturation antigen; B-cell maturation factor; B-cell maturation protein; tumor necrosis factor receptor superfamily member 17

(52) CT Ags—CTA (Cancer Testis Antigens) CROSS-REFERENCES

Fratta E., et al. Mol Oncol. 2011 April; 5(2):164-82; Lim S H., at al Am J Blood Res. 2012; 2(1):29-35.

(53) CD174 (Lewis Y)—FUT3 (Fucosyltransferase 3 (Galactoside 3(4)-L-Fucosyltransferase, Lewis Blood Group) Nucleotide

Genbank accession no. NM000149 Genbank version no. NM000149.3 GI:148277008 Genbank record update date: Jun. 26, 2012 04:49 PM

Polypeptide

Genbank accession no. NP_000140 Genbank version no. NP_000140.1 GI:4503809 Genbank record update date: Jun. 26, 2012 04:49 PM

CROSS-REFERENCES

Kukowska-Latallo, J. F., et al Genes Dev. 4 (8), 1288-1303 (1990)

Other Information Official Symbol: FUT3 Other Aliases: CD174, FT3B, FucT-III, LE, Les

Other Designations: Lewis F T; alpha-(1,3/1,4)-fucosyltransferase; blood group Lewis alpha-4-fucosyltransferase; fucosyltransferase III; galactoside 3(4)-L-fucosyltransferase (54) CLEC14A (C-Type Lectin Domain Family 14, Member a; Genbank Accession No. NM175060)

Nucleotide

Genbank accession no. NM175060 Genbank version no. NM175060.2 GI:371123930 Genbank record update date: Apr. 1, 2012 03:34 PM

Polypeptide

Genbank accession no. NP_778230 Genbank version no. NP_778230.1 GI:28269707 Genbank record update date: Apr. 1, 2012 03:34 PM

Other Information Official Symbol: CLEC14A Other Aliases: UNQ236/PRO269, C14orf27, CEG1, EGFR-5

Other Designations: C-type lectin domain family 14 member A; CIECT and EGF-like domain containing protein; epidermal growth factor receptor 5 (55) GRP78—HSPA5 (Heat Shock 70 kDa Protein 5 (Glucose-Regulated Protein, 78 kDa)

Nucleotide

Genbank accession no. NM005347 Genbank version no. NM005347.4 GI:305855105 Genbank record update date: Sep. 30, 2012 01:42 PM

Polypeptide

Genbank accession no. NP_005338 Genbank version no. NP_005338.1 GI:16507237 Genbank record update date: Sep. 30, 2012 01:42 PM

CROSS-REFERENCES

Ting J., et al DNA 7 (4), 275-286 (1988)

Other Information Official Symbol: HSPA5 Other Aliases: BIP, GRP78, MIF2

Other Designations: 78 kDa glucose-regulated protein; endoplasmic reticulum lumenal Ca(2+)-binding protein grp78; immunoglobulin heavy chain-binding protein

(56) Cd70 (Cd70 Molecule) L08096 Nucleotide

Genbank accession no. L08096 Genbank version no. L08096.1 GI:307127 Genbank record update date: Jun. 23, 2012 08:54 AM

Polypeptide

Genbank accession no. AAA36175 Genbank version no. AAA36175.1 GI:307128 Genbank record update date: Jun. 23, 2012 08:54 AM

CROSS-REFERENCES

Goodwin R. G., et al Cell 73 (3), 447-456 (1993)

Other Information Official Symbol: CD70 Other Aliases: CD27L, CD27LG, TNFSF7

Other Designations: CD27 ligand; CD27-L; CD70 antigen; Ki-24 antigen; surface antigen CD70; tumor necrosis factor (ligand) superfamily, member 7; tumor necrosis factor ligand superfamily member 7

Antibodies

MDX-1411 against CD70 (Medarex) h1F6 (Oflazoglu, E., et al, Clin Cancer Res. 2008 Oct. 1; 14(19):6171-80; Seattle Genetics)

-   -   For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and         FIG. 1.         (57) Stem Cell Specific Antigens. For Example:     -   5T4 (see entry (63) below)     -   CD25 (see entry (48) above)     -   CD32         -   Polypeptide             -   Genbank accession no. ABK42161             -   Genbank version no. ABK42161.1 GI:117616286             -   Genbank record update date: Jul. 25, 2007 03:00 PM         -   LGR5/GPR49         -   Nucleotide             -   Genbank accession no. NM_003667             -   Genbank version no. NM_003667.2 GI:24475886             -   Genbank record update date: Jul. 22, 2012 03:38 PM         -   Polypeptide             -   Genbank accession no. NP_003658             -   Genbank version no. NP_003658.1 GI:4504379             -   Genbank record update date: Jul. 22, 2012 03:38 PM     -   Prominin/CD133         -   Nucleotide             -   Genbank accession no. NM_006017             -   Genbank version no. NM_006017.2 GI:224994187             -   Genbank record update date: Sep. 30, 2012 01:47 PM         -   Polypeptide             -   Genbank accession no. NP_006008             -   Genbank version no. NP_006008.1 GI:5174387             -   Genbank record update date: Sep. 30, 2012 01:47 PM

(58) ASG-5 CROSS-REFERENCES

(Smith L. M., et. al AACR 2010 Annual Meeting (abstract #2590); Gudas J. M., et. al. AACR 2010 Annual Meeting (abstract #4393)

Antibodies

Anti-AGS-5 Antibody: M6.131 (Smith, L. M., et. al AACR 2010 Annual Meeting (abstract #2590)

(59) ENPP3 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 3) Nucleotide

Genbank accession no. AF005632 Genbank version no. AF005632.2 GI:4432589 Genbank record update date: Mar. 10, 2010 09:41 PM

Polypeptide

Genbank accession no. AAC51813 Genbank version no. AAC51813.1 GI:2465540 Genbank record update date: Mar. 10, 2010 09:41 PM

CROSS-REFERENCES

Jin-Hua P., et al Genomics 45 (2), 412-415 (1997)

Other Information Official Symbol: ENPP3 Other Aliases: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3

Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase I/nucleotide pyrophosphatase 3); dJ914N13.3 (phosphodiesterase I/nucleotide pyrophosphatase 3); ectonucleotide pyrophosphatase/phosphodiesterase family member 3; gp13ORB13-6; phosphodiesterase I beta; phosphodiesterase I/nucleotide pyrophosphatase 3; phosphodiesterase-I beta

(60) PRR4 (Proline Rich 4 (Lacrimal)) Nucleotide

Genbank accession no. NM_007244 Genbank version no. NM_007244.2 GI:154448885 Genbank record update date: Jun. 28, 2012 12:39 PM

Polypeptide

Genbank accession no. NP_009175 Genbank version no. NP_009175.2 GI:154448886 Genbank record update date: Jun. 28, 2012 12:39 PM

CROSS-REFERENCES

Dickinson D. P., et al Invest. Ophthalmol. Vis. Sci. 36 (10), 2020-2031 (1995)

Other Information Official Symbol: PRR4 Other Aliases: LPRP, PROL4

Other Designations: lacrimal proline-rich protein; nasopharyngeal carcinoma-associated proline-rich protein 4; proline-rich polypeptide 4; proline-rich protein 4

(61) GCC—GUCY2C (Guanylate Cyclase 2C (Heat Stable Enterotoxin Receptor) Nucleotide

Genbank accession no. NM_004963 Genbank version no. NM_004963.3 GI:222080082 Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_004954 Genbank version no. NP_004954.2 GI:222080083 Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS-REFERENCES

De Sauvage F. J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991); Singh S., et al Biochem. Biophys. Res. Commun. 179 (3), 1455-1463 (1991)

Other Information Official Symbol: GUCY2C Other Aliases: DIAR6, GUC2C, MUCIL, STAR

Other Designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR; heat-stable enterotoxin receptor; intestinal guanylate cyclase

(62) Liv-1—SLC39A6 (Solute Carrier Family 39 (Zinc Transporter), Member 6) Nucleotide

Genbank accession no. U41060 Genbank version no. U41060.2 GI:12711792 Genbank record update date: Nov. 30, 2009 04:35 PM

Polypeptide

Genbank accession no. AAA96258 Genbank version no. AAA96258.2 GI:12711793 Genbank record update date: Nov. 30, 2009 04:35 PM

CROSS-REFERENCES

Taylor K M., et al Biochim Biophys Acta. 2003 Apr. 1; 1611(1-2):16-30

Other Information Official Symbol: SLC39A6 Other Aliases: LIV-1

Other Designations: LIV-1 protein, estrogen regulated; ZIP-6; estrogen-regulated protein LIV-1; solute carrier family 39 (metal ion transporter), member 6; solute carrier family 39 member 6; zinc transporter ZIP6; zrt- and Irt-like protein 6

(63) 5T4, Trophoblast Glycoprotein, TPBG—TPBG (Trophoblast Glycoprotein) Nucleotide

Genbank accession no. AJ012159 Genbank version no. AJ012159.1 GI:3805946 Genbank record update date: Feb. 1, 2011 10:27 AM

Polypeptide

Genbank accession no. CAA09930 Genbank version no. CAA09930.1 GI:3805947 Genbank record update date: Feb. 1, 2011 10:27 AM

CROSS-REFERENCES

King K. W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999)

Other Information

-   -   Official Symbol: TPBG     -   Other Aliases: 5T4, 5T4AG, M6P1     -   Other Designations: 5T4 oncofetal antigen; 5T4 oncofetal         trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein

(64) CD56—NCMA1 (Neural Cell Adhesion Molecule 1) Nucleotide

Genbank accession no. NM_000615 Genbank version no. NM_000615.6 GI:336285433 Genbank record update date: Sep. 23, 2012 02:32 PM

Polypeptide

Genbank accession no. NP_000606 Genbank version no. NP_000606.3 GI:94420689 Genbank record update date: Sep. 23, 2012 02:32 PM

CROSS-REFERENCES

Dickson, G., et al, Cell 50 (7), 1119-1130 (1987)

Other Information Official Symbol: NCAM1 Other Aliases: CD56, MSK39, NCAM

Other Designations: antigen recognized by monoclonal antibody 5.1H11; neural cell adhesion molecule, NCAM

Antibodies Immunogen: HuN901 (Smith S V., et al Curr Opin Mol Ther. 2005 August; 7(4):394-401)

-   -   For example, see humanized from murine N901 antibody. See FIGS.         1b and 1e of Roguska, M. A., et al. Proc Natl Acad Sci USA         February 1994; 91:969-973.

(65) CanAg (Tumor Associated Antigen CA242) CROSS-REFERENCES

Haglund C., et al Br J Cancer 60:845-851, 1989; Baeckstrom D., et al J Biol Chem 266:21537-21547, 1991

Antibodies

huC242 (Tolcher A W et al., J Clin Oncol. 2003 Jan. 15; 21(2):211-22; Immunogen)

-   -   For example, see US20080138898A1 SEQ ID NO: 1 and 2

(66) FOLR1 (Folate Receptor 1) Nucleotide

Genbank accession no. J05013 Genbank version no. J05013.1 GI:182417 Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35823 Genbank version no. AAA35823.1 GI:182418 Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

Elwood P. C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989)

Other Information Official Symbol: FOLR1 Other Aliases: FBP, FOLR

Other Designations: FR-alpha; KB cells FBP; adult folate-binding protein; folate binding protein; folate receptor alpha; folate receptor, adult; ovarian tumor-associated antigen MOv18

Antibodies M9346A—Whiteman K R., et al Cancer Res Apr. 15, 2012; 72(8 Supplement): 4628 (Immunogen) (67) GPNMB (Glycoprotein (Transmembrane) Nmb) Nucleotide

Genbank accession no. X76534 Genbank version no. X76534.1 GI:666042 Genbank record update date: Feb. 2, 2011 10:10 AM

Polypeptide

Genbank accession no. CAA54044 Genbank version no. CAA54044.1 GI:666043 Genbank record update date: Feb. 2, 2011 10:10 AM

CROSS-REFERENCES

Weterman M. A., et al Int. J. Cancer 60 (1), 73-81 (1995)

Other Information Official Symbol: GPNMB Other Aliases: UNQ1725/PRO9925, HGFIN, NMB

Other Designations: glycoprotein NMB; glycoprotein nmb-like protein; osteoactivin; transmembrane glycoprotein HGFIN; transmembrane glycoprotein NMB

Antibodies Celldex Therapeutics: CR011 (Tse K F., et al Clin Cancer Res. 2006 Feb. 15; 12(4):1373-82)

-   -   For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and         35

(68) TIM-1—HAVCR1 (Hepatitis A Virus Cellular Receptor 1) Nucleotide

Genbank accession no. AF043724 Genbank version no. AF043724.1 GI:2827453 Genbank record update date: Mar. 10, 2010 06:24 PM

Polypeptide

Genbank accession no. AAC39862 Genbank version no. AAC39862.1 GI:2827454 Genbank record update date: Mar. 10, 2010 06:24 PM

CROSS-REFERENCES

Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998)

Other Information Official Symbol: HAVCR1 Other Aliases: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1

Other Designations: T cell immunoglobin domain and mucin domain protein 1; T-cell membrane protein 1; kidney injury molecule 1

(69) RG-1/Prostate Tumor Target Mindin—Mindin/RG-1 CROSS-REFERENCES

Parry R., et al Cancer Res. 2005 Sep. 15; 65(18):8397-405

(70) 87-H4—VTCN1 (V-Set Domain Containing T Cell Activation Inhibitor 1 Nucleotide

Genbank accession no. BX648021 Genbank version no. BX648021.1 GI:34367180 Genbank record update date: Feb. 2, 2011 08:40 AM

CROSS-REFERENCES

Sica G L., et al Immunity. 2003 June; 18(6):849-61

Other Information Official Symbol: VTCN1 Other Aliases: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PRO1291, VCTN1

Other Designations: B7 family member, H4; B7 superfamily member 1; T cell costimulatory molecule B7x; T-cell costimulatory molecule B7x; V-set domain-containing T-cell activation inhibitor 1; immune costimulatory protein B7-H4

(71) PTK7 (PTK7 Protein Tyrosine Kinase 7) Nucleotide

Genbank accession no. AF447176 Genbank version no. AF447176.1 GI:17432420 Genbank record update date: Nov. 28, 2008 01:51 PM

Polypeptide

Genbank accession no. AAL39062 Genbank version no. AAL39062.1 GI:17432421 Genbank record update date: Nov. 28, 2008 01:51 PM

CROSS-REFERENCES

Park S. K., et al J. Biochem. 119 (2), 235-239 (1996)

Other Information Official Symbol: PTK7 Other Aliases: CCK-4, CCK4

Other Designations: colon carcinoma kinase 4; inactive tyrosine-protein kinase 7; pseudo tyrosine kinase receptor 7; tyrosine-protein kinase-like 7

(72) Cd37 (Cd37 Molecule) Nucleotide

Genbank accession no. NM_001040031 Genbank version no. NM_001040031.1 GI:91807109 Genbank record update date: Jul. 29, 2012 02:08 PM

Polypeptide

Genbank accession no. NP_001035120 Genbank version no. NP_001035120.1 GI:91807110 Genbank record update date: Jul. 29, 2012 02:08 PM

CROSS-REFERENCES

Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988)

Other Information Official Symbol: CD37 Other Aliases: GP52-40, TSPAN26

Other Designations: CD37 antigen; cell differentiation antigen 37; leukocyte antigen CD37; leukocyte surface antigen CD37; tetraspanin-26; tspan-26

Antibodies Boehringer Ingelheim: mAb 37.1 (Heider K H., et al Blood. 2011 Oct. 13; 118(15):4159-68)

Trubion: CD37-SMIP (G28-1 scFv-Ig) ((Zhao X., et al Blood. 2007; 110: 2569-2577)

-   -   For example, see US20110171208A1 SEQ ID NO: 253

Immunogen: K7153A (Deckert J., et al Cancer Res Apr. 15, 2012; 72(8 Supplement): 4625) (73) CD138—SDC1 (Syndecan 1) Nucleotide

Genbank accession no. AJ551176 Genbank version no. AJ551176.1 GI:29243141 Genbank record update date: Feb. 1, 2011 12:09 PM

Polypeptide

Genbank accession no. CAD80245 Genbank version no. CAD80245.1 GI:29243142 Genbank record update date: Feb. 1, 2011 12:09 PM

CROSS-REFERENCES

O'Connell F P., et al Am J Clin Pathol. 2004 February; 121(2):254-63

Other Information Official Symbol: SDC1

Other Aliases: CD138, SDC, SYND1, syndecan Other Designations: CD138 antigen; heparan sulfate proteoglycan fibroblast growth factor receptor; syndecan proteoglycan 1; syndecan-1

Antibodies

Biotest: chimerized MAb (nBT062)—(Jagannath S., et al Poster ASH #3060, 2010; WIPO Patent Application WO/2010/128087)

-   -   For example, see US20090232810 SEQ ID NO: 1 and 2

Immunogen: B-B4 (Tassone P., et al Blood 104_3688-3696)

-   -   For example, see US20090175863A1 SEQ ID NO: 1 and 2

(74) CD74 (CD74 Molecule, Major Histocompatibility Complex, Class II Invariant Chain) Nucleotide

Genbank accession no. NM_004355 Genbank version no. NM_004355.1 GI:343403784 Genbank record update date: Sep. 23, 2012 02:30 PM

Polypeptide

Genbank accession no. NP_004346 Genbank version no. NP_004346.1 GI:10835071 Genbank record update date: Sep. 23, 2012 02:30 PM

CROSS-REFERENCES

Kudo, J., et al Nucleic Acids Res. 13 (24), 8827-8841 (1985)

Other Information Official Symbol: CD74 Other Aliases: DHLAG, HLADG, II, Ia-GAMMA

Other Designations: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); HLA class II histocompatibility antigen gamma chain; HLA-DR antigens-associated invariant chain; HLA-DR-gamma; Ia-associated invariant chain; MHC HLA-DR gamma chain; gamma chain of class II antigens; p33

Antibodies

Immunomedics: hLL1 (Milatuzumab,)—Berkova Z., et al Expert Opin Investig Drugs. 2010 January; 19(1):141-9)

-   -   For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23         and 24         Genmab: HuMax-CD74 (see website)

(75) Claudins—CLs (Claudins) CROSS-REFERENCES

Offner S., et al Cancer Immunol Immunother. 2005 May; 54(5):431-45, Suzuki H., et al Ann N Y Acad Sci. 2012 July; 1258:65-70)

In humans, 24 members of the family have been described—see literature reference.

(76) EGFR (Epidermal Growth Factor Receptor) Nucleotide

Genbank accession no. NM_005228 Genbank version no. NM_005228.3 GI:41927737 Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005219 Genbank version no. NP_005219.2 GI:29725609 Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

Dhomen N S., et al Crit Rev Oncog. 2012; 17(1):31-50

Other Information Official Symbol: EGFR

Other Aliases: ERBB, ERBB1, HER1, PIG61, mENA Other Designations: avian erythroblastic leukemia viral (v-erb-b) oncogene homolog; cell growth inhibiting protein 40; cell proliferation-inducing protein 61; proto-oncogene c-ErbB-1; receptor tyrosine-protein kinase erbB-1

Antibodies BMS: Cetuximab (Erbitux)—Broadbridge V T., et al Expert Rev Anticancer Ther. 2012 May; 12(5):555-65.

-   -   For example, see U.S. Pat. No. 6,217,866—ATTC deposit No. 9764.

Amgen: Panitumumab (Vectibix)—Argiles G., et al Future Oncol. 2012 April; 8(4):373-89

-   -   For example, see U.S. Pat. No. 6,235,883 SEQ ID NOs: 23-38.

Genmab: Zalutumumab—Rivera F., et al Expert Opin Biol Ther. 2009 May; 9(5):667-74. YM Biosciences: Nimotuzumab—Ramakrishnan M S., et al MAbs. 2009 January-February; 1(1):41-8.

-   -   For example, see U.S. Pat. No. 5,891,996 SEQ ID NOs: 27-34.         (77) Her3 (ErbB3)—ERBB3 (v-erb-b2 erythroblastic leukemia viral         oncogene homolog 3 (avian))

Nucleotide

Genbank accession no. M34309 Genbank version no. M34309.1 GI:183990 Genbank record update date: Jun. 23, 2010 08:47 PM

Polypeptide

Genbank accession no. AAA35979 Genbank version no. AAA35979.1 GI:306841 Genbank record update date: Jun. 23, 2010 08:47 PM

CROSS-REFERENCES

Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13), 4905-4909 (1990)

Other Information Official Symbol: ERBB3

Other Aliases: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3, erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3 Other Designations: proto-oncogene-like protein c-ErbB-3; receptor tyrosine-protein kinase erbB-3; tyrosine kinase-type cell surface receptor HER3

Antibodies Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res. 2010 Mar. 15; 70(6):2485-2494)

-   -   For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7         and 8.         (78) RON—MST1R (Macrophage Stimulating 1 Receptor (c-Met-Related         Tyrosine Kinase))

Nucleotide

Genbank accession no. X70040 Genbank version no. X70040.1 GI:36109 Genbank record update date: Feb. 2, 2011 10:17 PM

Polypeptide

Genbank accession no. CCA49634 Genbank version no. CCA49634.1 GI:36110 Genbank record update date: Feb. 2, 2011 10:17 PM

CROSS-REFERENCES

Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993)

Other Information Official Symbol: MST1R Other Aliases: CD136, CDw136, PTK8, RON

Other Designations: MSP receptor; MST1R variant RON30; MST1R variant RON62; PTK8 protein tyrosine kinase 8; RON variant E2E3; c-met-related tyrosine kinase; macrophage-stimulating protein receptor; p185-Ron; soluble RON variant 1; soluble RON variant 2; soluble RON variant 3; soluble RONvariant 4

(79) EPHA2 (EPH Receptor A2) Nucleotide

Genbank accession no. BC037166 Genbank version no. BC037166.2 GI:33879863 Genbank record update date: Mar. 6, 2012 01:59 PM

Polypeptide

Genbank accession no. AAH37166 Genbank version no. AAH37166.1 GI:22713539 Genbank record update date: Mar. 6, 2012 01:59 PM

CROSS-REFERENCES

Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002)

Other Information Official Symbol: EPHA2 Other Aliases: ARCC2, CTPA, CTPP1, ECK

Other Designations: ephrin type-A receptor 2; epithelial cell receptor protein tyrosine kinase; soluble EPHA2 variant 1; tyrosine-protein kinase receptor ECK

Antibodies Medimmune: 1C1 (Lee J W., et al Clin Cancer Res. 2010 May 1; 16(9):2562-2570)

-   -   For example, see US20090304721A1 FIGS. 7 and 8.

(80) CD20—MS4A1 (Membrane-Spanning 4-Domains, Subfamily a, Member 1) Nucleotide

Genbank accession no. M27394 Genbank version no. M27394.1 GI:179307 Genbank record update date: Nov. 30, 2009 11:16 AM

Polypeptide

Genbank accession no. AAA35581 Genbank version no. AAA35581.1 GI:179308 Genbank record update date: Nov. 30, 2009 11:16 AM

CROSS-REFERENCES

Tedder T. F., et al Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212 (1988)

Other Information Official Symbol: MS4A1 Other Aliases: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7

Other Designations: B-lymphocyte antigen CD20; B-lymphocyte cell-surface antigen B1; CD20 antigen; CD20 receptor; leukocyte surface antigen Leu-16

Antibodies Genentech/Roche: Rituximab—Abdulla N E., et al BioDrugs. 2012 Apr. 1; 26(2):71-82.

-   -   For example, see U.S. Pat. No. 5,736,137, ATCC deposit No.         HB-69119.

GSK/Genmab: Ofatumumab—Nightingale G., et al Ann Pharmacother. 2011 October; 45(10):1248-55.

-   -   For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5.

Immunomedics: Veltuzumab—Goldenberg D M., et al Leuk Lymphoma. 2010 May; 51(5):747-55.

-   -   For example, see U.S. Pat. No. 7,919,273B2 SEQ ID NOs: 1, 2, 3,         4, 5 and 6.

(81) Tenascin C—TNC (Tenascin C) Nucleotide

Genbank accession no. NM_002160 Genbank version no. NM_002160.3 GI:340745336 Genbank record update date: Sep. 23, 2012 02:33 PM

Polypeptide

Genbank accession no. NP_002151 Genbank version no. NP_002151.2 GI:153946395 Genbank record update date: Sep. 23, 2012 02:33 PM

CROSS-REFERENCES

Nies D. E., et al J. Biol. Chem. 266 (5), 2818-2823 (1991); Sin A., et al Nucleic Acids Res. 19 (3), 525-531 (1991)

Other Information Official Symbol: TNC Other Aliases: 150-225, GMEM, GP, HXB, JI, TN, TN-C

Other Designations: GP 150-225; cytotactin; glioma-associated-extracellular matrix antigen; hexabrachion (tenascin); myotendinous antigen; neuronectin; tenascin; tenascin-C isoform 14/AD1/16

Antibodies Philogen: G11 (von Lukowicz T., et al J Nucl Med. 2007 April; 48(4):582-7) and F16 (Pedretti M., et al Lung Cancer. 2009 April; 64(1):28-33)

-   -   For example, see U.S. Pat. No. 7,968,685 SEQ ID NOs: 29, 35, 45         and 47.

(82) FAP (Fibroblast Activation Protein, Alpha) Nucleotide

Genbank accession no. U09278 Genbank version no. U09278.1 GI:1888315 Genbank record update date: Jun. 23, 2010 09:22 AM

Polypeptide

Genbank accession no. AAB49652 Genbank version no. AAB49652.1 GI:1888316 Genbank record update date: Jun. 23, 2010 09:22 AM

CROSS-REFERENCES

Scanlan, M. J., et al Proc. Natl. Acad. Sci. U.S.A. 91 (12), 5657-5661 (1994)

Other Information Official Symbol: FAP Other Aliases: DPPIV, FAPA

Other Designations: 170 kDa melanoma membrane-bound gelatinase; integral membrane serine protease; seprase (83) DKK-1 (Dickkopf 1 Homolog (Xenopus laevis)

Nucleotide

Genbank accession no. NM_012242 Genbank version no. NM_012242.2 GI:61676924 Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_036374 Genbank version no. NP_036374.1 GI:7110719 Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999)

Other Information Official Symbol: DKK1 Other Aliases: UNQ492/PRO1008, DKK-1, SK

Other Designations: dickkopf related protein-1; dickkopf-1 like; dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1

Antibodies Novartis: BHQ880 (Fulciniti M., et al Blood. 2009 Jul. 9; 114(2):371-379)

-   -   For example, see US20120052070A1 SEQ ID NOs: 100 and 108.

(84) Cd52 (Cd52 Molecule) Nucleotide

Genbank accession no. NM_001803 Genbank version no. NM_001803.2 GI:68342029 Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001794 Genbank version no. NP_001794.2 GI:68342030 Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

Xia M. Q., et al Eur. J. Immunol. 21 (7), 1677-1684 (1991)

Other Information Official Symbol: CD52 Other Aliases: CDW52

Other Designations: CAMPATH-1 antigen; CD52 antigen (CAMPATH-1 antigen); CDW52 antigen (CAMPATH-1 antigen); cambridge pathology 1 antigen; epididymal secretory protein E5; he5; human epididymis-specific protein 5

Antibodies Alemtuzumab (Campath)—Skoetz N., et al Cochrane Database Syst Rev. 2012 Feb. 15; 2:CD008078.

-   -   For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTD00109)

(85) CS1-SLAMF7 (SLAM Family Member 7) Nucleotide

Genbank accession no. NM_021181 Genbank version no. NM_021181.3 GI:1993571 Genbank record update date: Jun. 29, 2012 11:24 AM

Polypeptide

Genbank accession no. NP_067004 Genbank version no. NP_067004.3 GI:19923572 Genbank record update date: Jun. 29, 2012 11:24 AM

CROSS-REFERENCES

Boles K. S., et al Immunogenetics 52 (3-4), 302-307 (2001)

Other Information Official Symbol: SLAMF7 Other Aliases: UNQ576/PRO1138, 19A, CD319, CRACC, CS1

Other Designations: 19A24 protein; CD2 subset 1; CD2-like receptor activating cytotoxic cells; CD2-like receptor-activating cytotoxic cells; membrane protein FOAP-12; novel LY9 (lymphocyte antigen 9) like protein; protein 19A

Antibodies

BMS: elotuzumab/HuLuc63 (Benson D M., et al J Clin Oncol. 2012 Jun. 1; 30(16):2013-2015)

-   -   For example, see US20110206701 SEQ ID NOs: 9, 10, 11, 12, 13,         14, 15 and 16.

(86) Endoglin—ENG (Endoglin) Nucleotide

Genbank accession no. AF035753 Genbank version no. AF035753.1 GI:3452260 Genbank record update date: Mar. 10, 2010 06:36 PM

Polypeptide

Genbank accession no. AAC32802 Genbank version no. AAC32802.1 GI:3452261 Genbank record update date: Mar. 10, 2010 06:36 PM

CROSS-REFERENCES

Rius C., et al Blood 92 (12), 4677-4690 (1998)

Official Symbol: ENG Other Information Other Aliases: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1

Other Designations: CD105 antigen

(87) Annexin A1—ANXA1 (Annexin A1) Nucleotide

Genbank accession no. X05908 Genbank version no. X05908.1 GI:34387 Genbank record update date: Feb. 2, 2011 10:02 AM

Polypeptide

Genbank accession no. CCA29338 Genbank version no. CCA29338.1 GI:34388 Genbank record update date: Feb. 2, 2011 10:02 AM

CROSS-REFERENCES

Wallner B. P., et al Nature 320 (6057), 77-81 (1986)

Other Information Official Symbol: ANXA1 Other Aliases: RP11-71A24.1, ANX1, LPC1

Other Designations: annexin I (lipocortin I); annexin-1; calpactin II; calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2 inhibitory protein

(88) V-CAM (CD106)—VCAM1 (Vascular Cell Adhesion Molecule 1) Nucleotide

Genbank accession no. M60335 Genbank version no. M60335.1 GI:340193 Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA61269 Genbank version no. AAA61269.1 GI:340194 Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS-REFERENCES

Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991)

Other Information Official Symbol VCAM1 Other Aliases: CD106, INCAM-100

Other Designations: CD106 antigen; vascular cell adhesion protein 1

Antibody Sequences Anti-Integrin αvβ6 RHAB6.2 QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDT EYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVA GPYPFDYWGQGTLVTVSS RHCB6.2 QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDT EYAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKV AGPYPFDYWGQGTLVTVSS RHF QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGD TEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLV TVSS RHFB6 QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGD TEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQK VAGPYYFDYWGQGTLVTVSS RHAY100bP QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDT EYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTV SS RKF ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKFL36L50 ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKC EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK Anti-CD33 CD33 Hum195 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGT GYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS CD33 Hum195 VK DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK Anti-CD19 CD19 B4 resurfaced VH QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYT NYNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSV TVSS CD19 B4 resurfaced VK EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPA RFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK Anti-Her2 Herceptin VH chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTR YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSS Herceptin VL chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Anti-CD25 Simulect VK (also known as Basiliximab) QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK Simulect VH QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSY NQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS Anti-PSMA Deimmunised VH ′1 EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTY NQKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS Deimmunised VK ′1 DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPS RFSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK Deimmunised VH1 ′5 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNN FATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH2 ′5 EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNF ATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH3 ′5 EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNF ATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH4 ′5 EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNF ATHYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VK1 ′5 NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVP DRFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK Deimmunised VK2 ′5 NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVP DRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK3 ′5 NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVP DRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK4 ′5 NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVP DRFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK DI ′5 NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVP DRFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK Deimmunised VH DI ′5 EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNF ATHYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHA ′5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNF ATHYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHB ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNF ATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHC ′5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNF ATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHD ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNF ATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHE ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNF ATHYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHF ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNF ATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHG ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNF ATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RKA ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKB ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKC ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKD ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKE ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPD RFTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKF ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKG ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPD RFTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (Dennis et al. (2002) “Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of the invention include fusion proteins with ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 at pages 12-13, and all of which are incorporated herein by reference.

In one embodiment, the antibody has been raised to target specific the tumour related antigen α_(v)β₆.

The cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be labelled with a radioisotope.

Connection of Linker Unit to Ligand Unit

The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit.

In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond.

In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond.

In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond.

In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond.

In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit.

The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.

In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein.

Methods of Treatment

The compounds of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound of formula I. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

A compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

The Compounds and Conjugates can be used to treat proliferative disease and autoimmune disease. The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.

Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose.

Drug Loading

The drug loading is the average number of PBD drugs per cell binding agent, e.g. antibody. Where the compounds of the invention are bound to cysteines, drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8. Where the compounds of the invention are bound to lysines, drug loading may range from 1 to 80 drugs (D) per cell binding agent, although an upper limit of 40, 20, 10 or 8 may be preferred. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA. Also, ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p>5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541 teaches engineering antibodies by introduction of reactive cysteine amino acids.

Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249). The engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties. The location of the drug moiety can thus be designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibody reacts with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value. Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.

Thus the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group per cell binding agent.

Includes Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O⁻), a salt or solvate thereof, as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Carbinolamines

The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example, by lyophilisation.

Isomers

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C₁₋₇ alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and³H (T); C may be in any isotopic form, including ¹²O, ¹³O, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:

a) WO 00/12508 (pages 14 to 30); b) WO 2005/023814 (pages 3 to 10); c) WO 2004/043963 (pages 28 to 29); and d) WO 2005/085251 (pages 30 to 39).

Synthesis Route

The compounds of the present invention, where R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Formula 2:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R¹², X, X′ and R″ are as defined for compounds of formula I, Prot^(N) is a nitrogen protecting group for synthesis and Prot^(O) is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods.

The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if Prot^(N) is Alloc and Prot^(O) is an oxygen protecting group for synthesis, then the deprotection is carried using palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for synthesis. If Prot^(N) is Troc and Prot^(O) is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If Prot^(N) is SEM, or an analogous group, and Prot^(O) is an an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Formula 2 can be accomplished by, for example, lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel.

Compounds of formula 2 can be synthesised from a compound of formula 3:

where R¹², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R², such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.

The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂, Pd₂(dba)₃. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.

Compounds of formula I where R¹⁰ and R^(10′) are H and R¹¹ and R^(11′) are SO_(z)M, can be synthesised from compounds of formula I where R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.

The PBD monomer components can be synthesised and linked in a similar manner to that described in WO 2005/085259, where one monomer unit is linked to the tether unit, following by reaction with a second monomer unit.

Nitrogen Protecting Groups for Synthesis

Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups.

Carbamate nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.

Other possible groups are nitrobenzyloxycarbonyl (e.g. 4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.

Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc.

Hemi-aminal nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds of the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, Cl₃CCH₂OCH₂—.

Protected Oxygen Group for Synthesis

Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.

Preferred oxygen protecting groups include acetates, TBS and THP.

Synthesis of Drug Conjugates

Conjugates can be prepared as previously described. Linkers having a maleimidyl group (A), a peptide group (L¹) and self-immolative group (L²) can be prepared as described in U.S. Pat. No. 6,214,345. Linkers having a maleimidyl group (A) and a peptide group (L¹) can be prepared as described in WO 2009-0117531. Other linkers can be prepared according to the references cited herein or as known to the skilled artisan.

Linker-Drug compounds can be prepared according to methods known in the art. Linkage of amine-based X substituents (of the PDB dimer Drug unit) to active groups of the Linker units can be performed according to methods generally described in U.S. Pat. Nos. 6,214,345 and 7,498,298; and WO 2009-0117531, or as otherwise known to the skilled artisan.

Antibodies can be conjugated to Linker-Drug compounds as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC-quenched drug-linker.

Further Preferences

The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), R^(10′), R^(11′), and Y′ are preferably the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹, and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C₃₋₇ alkylene group with no substituents. More preferably R″ is a C₃, C₅ or C₇ alkylene. Most preferably, R″ is a C₃ or C₅ alkylene.

R⁶ to R⁹

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, and is more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C₁₋₇ alkyl, C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably a C₁₋₄ alkyl group, which may or may not be substituted. A substituent of interest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH₂Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe₂); —(OC₂H₄)_(q)OMe, where q is from 0 to 2; nitrogen-containing C₆ heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.

These preferences apply to R^(9′), R^(6′) and R^(7′) respectively.

R²

A in R² may be phenyl group or a C₅₋₇ heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl.

X is a group selected from the list comprising: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

and NHR^(N), wherein R^(N) is selected from the group comprising H and C₁₋₄ alkyl. X may preferably be: OH, SH, CO₂H, —N═C═O or NHR^(N), and may more preferably be: OH, SH, CO₂H, —N═C═O or NH₂. Particularly preferred groups include: OH, SH and NH₂, with NH₂ being the most preferred group.

Q²-X may be on any of the available ring atoms of the C₅₋₇ aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C₅₋₇ aryl group (A) is phenyl, the substituent (Q²-X) is preferably in the meta- or para-positions, and more preferably is in the para-position.

In some embodiments, Q¹ is a single bond. In these embodiments, Q² is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q² is a single bond. In other embodiments, Q² is —Z—(CH₂)_(n)—. In these embodiments, Z may be O or S and n may be 1 or n may be 2. In other of these embodiments, Z may be a single bond and n may be 1.

In other embodiments, Q¹ is —CH═CH—.

In some embodiments, R² may be -A-CH₂—X and -A-X. In these embodiments, X may be OH, SH, CO₂H, COH and NH₂. In particularly preferred embodiments, X may be NH₂.

R¹²

In one embodiment, R¹² is ═CH₂.

In one embodiment, R¹² is ═CH—R^(D1). Within the PBD compound, the group ═CH—R^(D1) may have either configuration shown below:

In one embodiment, the configuration is configuration (I).

R^(D1) and R^(D2) (when present) may be selected from R, and in particular from C₁₋₃ alkyl, e.g. methyl, ethyl, propyl. In some of these embodiments, R^(D1) and R^(D2) (when present) are methyl.

R¹⁰ and R¹¹

In one embodiment, R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl. In some of these embodiments, R^(A) is methyl.

In another embodiment, R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

In another embodiment, R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.

M and z

It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na⁺.

z is preferably 3.

Particularly preferred compounds of the present invention are of formula Ia:

where R^(1a) is selected from Me and Ph, and the amino group is at either the meta or para positions of the phenyl group.

3^(rd) Aspect

The preferences expressed above for the first aspect may apply to the compounds of this aspect, where appropriate.

When R¹⁰ is carbamate nitrogen protecting group, it may preferably be Teoc, Fmoc and Troc, and may more preferably be Troc.

When R¹¹ is O-Prot^(O), wherein Prot^(O) is an oxygen protecting group, Prot^(O) may preferably be TBS or THP, and may more preferably be TBS.

When R¹⁰ is a hemi-aminal nitrogen protecting group, it may preferably be MOM, BOM or SEM, and may more preferably be SEM.

The preferences for compounds of formula I apply as appropriate to D in the sixth aspect of the invention.

EXAMPLES General Experimental Methods

Reaction progress was monitored by thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. Extraction and chromatography solvents were bought and used without further purification from Fisher Scientific, U.K. All chemicals were purchased from Aldrich, Lancaster or BDH.

Method for LS/MS Spectrometer:

LC/MS (Shimazu LCMS-2020) using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 0.25 min, then increase from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutes and hold there for 0.05 min. Total gradient run time equals 3 min. Flow rate 0.8 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50° C. Column: Waters Acquity UPLC BEH Shield RP18 1.7 μm 2.1×50 mm.

Method for Preparative HPLC:

HPLC (Shimadzu UFLC) was run using a mobile phase of water (0.1% formic acid) A and acetonitrile (0.1% formic acid) B.

Wavelength detection range: 254 nm.

Column: Phenomenex Gemini 5μ C18 150×21-20 mm.

Gradient:

B t = 0 13% t = 15.00 95% t = 17.00 95% t = 17.10 13% t = 20.00 13%

Total gradient run time is 20 min; flow rate 20.00 mL/min.

Example 1 (i) tert-Butyl (11S,11aS)-2-(4-(2-((R)-1-(((S)-1-((9H-fluoren-9-yl)methoxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)phenyl)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (10)

(a) (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (2)

Ketone monomer 1 (20 g, 34 mmol, 1 eq.) was solubilised in dry CH₂Cl₂ (350 mL), before 2,6-lutidine (16.06 mL, 0.137 mol, 4 eq.) was added and the mixture cooled to −50° C. Triflic anhydride (17.37 mL, 0.1 mol, 3 eq) was then added dropwise maintaining the temperature below −40° C. When the reaction was completed, excess Tf₂O was quenched with H₂O (250 mL). The layers were separated and the organics were washed further with aqueous NaHCO_(3 (sat.)) (200 mL) and brine (200 mL). The crude mixture was purified by silica gel chromatography (gradient elution: 100% hexane to 90:10 v/v hexane/EtOAc) to afford pure product 2 as brown foam (22.065 g, 89% yield). Analytical data: ES⁺=2.39 min, m/z 1448.20 [2M+Na]⁺.

(b) (S)-(4-(4-aminophenyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (3)

Pd(PPh₃)₄ (609 mg, 520 mmol, 0.02 eq.) was added to a stirred mixture of triflate 2 (18.8 g, 26.3 mmol, 1 eq.), 4-aminophenylboronic acid pinacol ester (8.64 g, 39.4 mmol, 1.5 eq) and Na₂CO₃ (12.7 g, 120 mmol, 4.6 eq.) in MeOH (75 mL), toluene (150 mL) and water (75 mL). The reaction mixture was allowed to stir at 30° C. under an argon atmosphere for 24 hours after which time all the triflate has been consumed. The reaction mixture was then evaporated to dryness before the residue was taken up in EtOAc (400 mL) and washed with H₂O (2×250 mL), brine (250 mL), dried (MgSO₄), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 100% hexane to 80:20 v/v hexane/EtOAc) afforded product 3 as a yellowish foam (11.058 g, 64% yield). Analytical data: ES⁺=2.20 min, m/z not observed.

(c) (9H-fluoren-9-yl)methyl (S)-(4-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl)phenyl)carbamate (4)

To a dry round bottom flask was added aniline 3 (10.05 g, 15.3 mmol), Fmoc-Val-Ala-OH (6.3 g, 15.3 mmol) and dry CH₂Cl₂ (500 mL). The flask was then purged 3 times with argon before EEDQ (3.79 mg, 15.3 mmol) was added and the mixture left to stir at room temperature. The reaction was followed by LCMS and left to stir for 16 hours. The reaction was quenched with H₂O (300 mL). The separated organics were washed with brine (250 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (gradient elution 80:20 v/v hexane/EtOAc to 50:50 v/v hexane/EtOAc) to afford pure product 4 (13.821 g, 86% yield). Analytical data: ES⁺=2.37 min, m/z 1071.65 [M+Na]⁺.

(d) (9H-fluoren-9-yl)methyl (S)-(4-(1-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl)phenyl)carbamate (5)

Monomer 4 (16.821 g, 16.04 mmol, 1 eq.) was solubilised in a mixture of methanol (600 mL) and formic acid (30 mL). Zinc dust was added slowly (10.49, 160 mmol, 10 eq.) and an exotherm was observed (temperature went up to 30° C.). After 25 minutes the reaction looked complete by TLC (eluent 1:1 v/v CH₂Cl₂/Et₂O). The reaction mixture was filtered through celite, the pad was washed with EtOAc (200 mL) and the solvent removed into vacuo. The dried crude was taken up in EtOAc (300 mL) and washed with H₂O (200 mL), NaHCO₃ (200 mL) and brine (200 mL), dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (elution 2:1 v/v hexane/EtOAc) to afford pure product 5 (12.642 g, 77% yield). Analytical data: ES⁺=2.36 min, m/z 1018.35 [M+H]⁺.

(e) (9H-fluoren-9-yl)methyl (S)-(4-(1-(2-((tert-butoxycarbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl)phenyl)carbamate (6)

Monomer 5 (14.913 g, 14.6 mmol, 1 eq.), together with Boc anhydride (3.83 g, 17.5 mmol, 1.2 eq.) was heated to 70° C., 25 mL of CH₂Cl₂ were also added to help solubilising the starting material and was subsequently left to evaporate with heating. The reaction was complete after 2 hours. The reaction mixture was put straight away on silica gel column chromatography (gradient elution: 100% hexane to 65:35 v/v hexane/EtOAc) to give pure product 6 as a light yellow foam (13.2 g, 80% yield). Analytical data: ES⁺=2.53 min, m/z 1118.35 [M+H]⁺.

(f) (9H-fluoren-9-yl)methyl (S)-(4-(1-(2-((tert-butoxycarbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrol-3-yl)phenyl)carbamate (7)

Monomer 6 (13.2 g, 13.9 mmol) was solubilised in a mixture of 7:1:1:2 v/v of AcOH/MeOH/THF/H₂O and left to stir for 16 hours. The solvents were subsequently removed and the crude taken up in EtOAc (300 mL), washed with H₂O (200 mL), NaHCO₃ (2×200 mL) and brine (150 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% hexane to 100% EtOAc) to afford pure product 7 (11.168 g, 94% yield).

Analytical data: ES⁺=2.23 min, m/z not observed.

(g) tert-Butyl (11S,11aS)-2-(4-(2-((R)-1-(((S)-1-((9H-fluoren-9-yl)methoxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)phenyl)-11-hydroxy-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (8)

In a dry flask purged with argon, dry DMSO (1.55 mL, 21.9 mmol, 2.5 eq.) was added to a solution of oxaly chloride (0.89 mL, 10.5 mmol, 1.2 eq.) in dry CH₂Cl₂ (50 mL) at −78° C. The mixture was left to stir for 20 minutes before monomer 7 (8.8 g, 8.76 mmol, 1 eq.) in CH₂Cl₂ (100 mL) was added. The mixture was left to stir for 1.5 hours at −78° C. before TEA (6.11 mL, 43.8 mmol, 5 eq.) was added and the mixture left to warm to room temperature.

After another 2 hours the reaction was quenched with 0.1M aqueous HCl (100 mL). The layers were separated and the organics were washed further with H₂O (150 mL) and brine (150 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% hexane to 1:1:0.1 hexane/EtOAc/MeOH) to afford a 6:4 mixture of product 7 and opened aldehyde monomer. That mixture was stirred overnight in CHCl₃ to give 100% cyclised product 7 (8.8 g, 100% yield). Analytical data: ES⁺=2.19 min, m/z not observed.

(h) tert-Butyl (11S,11aS)-2-(4-(2-((R)-1-(((S)-1-((9H-fluoren-9-yl)methoxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)phenyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (9)

Monomer 8 (8.8 g, 8.78 mmol, 1 eq.) was solubilised in CH₂Cl₂ (200 mL). The mixture was cooled to 0° C. before slowly adding 2,6-lutidine (4 mL, 35.1 mmol, 4 eq.) and TBS-OTf (6 mL, 26.3 mmol, 3 eq.). The mixture was subsequently left to warm to room temperature. Once the reaction was complete, the mixture was washed with aqueous NH₄Cl_((sat.)) (150 mL), H₂O (150 mL), aqueous NaHCO_(3 (sat.)) (150 mL) and brine (150 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% hexane to 6:4 v/v hexane/EtOAc) to afford pure product 9 (6.18 g, 70% yield). Analytical data: ES⁺=2.53 min, m/z not observed.

(i) tert-Butyl (11S,11aS)-2-(4-(2-((R)-1-(((S)-1-((9H-fluoren-9-yl)methoxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)phenyl)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (10)

Monomer 9 (3.1 g, 2.84 mmol, 1 eq.) was solubilised in DMF (8 mL) and H₂O (0.2 mL). LiOAc.H₂O (290 mg, 2.84 mmol, 1 eq.) was added. Then as much water as possible was added to the mixture without making the starting material crash out (3.5 mL). Once complete (≈1.5 hours) the reaction was quenched by aqueous citric acid (pH=3, 50 mL) and extracted with EtOAc (3×150 mL). The combined organics were washed with H₂O (150 mL) and brine (100 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 6:4 v/v hexane/EtOAc to 6:4:1 v/v hexane/EtOAc/MeOH) to afford pure product 10 (2.219 g, 83% yield). Analytical data: ES⁺=1.91 min, m/z 960.55 [M+H]⁺.

(ii) 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((2S)-1-(((2S)-1-((4-(7-methoxy-8-((5-((7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (15)

Compound 11 is compound 27 in WO 2005/085259.

(a) tert-Butyl (11S,11aS)-8-((5-iodopentyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (12)

Monomer 11 (200 mg, 0.43 mmol, 1 eq.) was solubilised in DMF (4 mL). 1,5-Diiodopentate (320 μL, 2.17 mmol, 5 eq.) and K₂CO₃ (93 mg, 0.75 mmol, 1 eq.) were added and the mixture heated to 60° C. Upon completion the mixture was diluted with EtOAc (100 mL) and washed with H₂O (2×50 mL) and brine (50 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% hexane to 7:3 v/v hexane/EtOAc) to afford pure product 12 (252.2 mg, 88% yield). Analytical data: ES⁺=1.95 min, m/z 657.25 [M+H]⁺.

(b) tert-Butyl (11S,11aS)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (13)

Monomer 10 (403 mg, 0.42 mmol, 1.1 eq.) and monomer 12 (250 mg, 0.38 mmol, 1 eq.) were solubilised in dry DMF (5 mL) under argon. K₂CO₃ (80.8 mg, 0.38 mmol, 1 eq.) was added and the mixture heated to 60° C. Upon completion the mixture was diluted with EtOAc (100 mL) and washed with H₂O (2×50 mL) and brine (50 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% CHCl₃ to 9:1 v/v CHCl₃/MeOH) to afford pure product 13 (260.9 mg, 54% yield). Analytical data: ES⁺=1.60 min, m/z 1267.25 [M+H]⁺.

(c) tert-Butyl (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-2-(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)phenyl)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (14)

EDCl hydrochloride (39 mg, 0.197 mmol, 1 eq.) was added to a suspension of maleimide-PEG₈-acid (117 mg, 0.197 mmol, 1 eq.) in dry CH₂Cl₂ (5 mL) under argon atmosphere. The mixture was stirred for 30 min at room temperature before PBD 13 (250 mg, 0.197 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH₂Cl₂ and the organic phase was washed with H₂O and brine before being dried over MgSO₄, filtered and excess solvent removed by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (gradient elution: 100% CHCl₃ to 9:1 v/v CHCl₃/MeOH) to afford pure product 14 (273.8 mg, 75% yield). Analytical data: ES⁺=1.99 min, m/z 1863.95 [M+Na]⁺.

(d) 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((2S)-1-(((2S)-1-((4-(7-methoxy-8-((5-((7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (15)

To PBD 14 (260 mg, 0.14 mmol, 1 eq.) was added H₂O (80 μL) and the slurry was cooled to 0° C. TFA (2 mL) was then added and the mixture was left to stir at 0° C. until completion. The reaction was subsequently quenched with aqueous NaHCO_(3 (sat.)) and extracted with CH₂Cl₂ (2×50 mL). The combined organics were then washed with H₂O (50 mL) and brine (50 mL) before being dried (MgSO₄), filtered and evaporated under reduced pressure. The crude was purified by preparative HPLC and the product fractions combined and freeze dried on the freeze drier overnight to afford pure product 15 (29.9 mg, 16% yield). Analytical data: ES⁺=1.41 min, m/z 1407.25 [M+H]⁺.

Example 2 Conjugation A

Trastuzumab (10 mg, 66.7 nanomoles) was diluted into 9.0 mL of a reduction buffer containing 10 mM sodium borate pH 8.4, 2.5 mM EDTA and a final antibody concentration of 1.11 mg/mL. A 10 mM solution of TCEP was added (1.7 molar equivalent/antibody, 113.3 nanomoles, 11.33 μL) and the reduction mixture was heated at +37° C. for 1.5 hours in an incubator with 150 rpm shaking. After cooling down to room temperature, compound 14 was added as a DMSO solution (7.5 molar equivalent/antibody, 525 nanomoles, in 1.0 mL DMSO). The solution was mixed for 1.5 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (1 micromole, 100 μL at 10 mM), then injected into an AKTA™ Pure FPLC using a GE Healthcare HiLoad™ 26/600 column packed with Superdex 200 PG, eluting with 2.6 mL/min of sterile-filtered phosphate-buffered saline (PBS). Fractions corresponding to ConjA monomer peak were pooled, concentrated using a 15 mL Amicon Ultracell 50KDa MWCO spin filter, analysed and sterile-filtered.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjA at 280 nm and 330 nm (Compound 14 specific) shows a mixture of light and heavy chains attached to several molecules of compound 14, consistent with a drug-per-antibody ratio (DAR) of 2.59 molecules of compound 14 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Yarra 3u SEC-3000 300 mm×4.60 mm eluting with sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomer purity of over 97% with no impurity detected. UHPLC SEC analysis gives a concentration of final ConjA at 1.59 mg/mL in 3.8 mL, obtained mass of ConjA is 6.04 mg (60% yield).

Conjugation B

A 50 mM solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (50 molar equivalent/antibody, 35 micromoles, 700 μL) to a 24.14 mL solution of antibody (105 mg, 700 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.35 mg/mL. The reduction mixture was heated at +37° C. for 3 hours (or until full reduction is observed by UHPLC) in an incubator with gentle (<150 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 10 molar equivalent/antibody, 7 micromoles, 140 μL) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (<150 rpm) shaking at an antibody concentration of 2.3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS pH 7.4, 1 mM EDTA for a final antibody concentration of 1.0-1.5 mg/mL. Compound 14 was added as a DMSO solution (10 molar equivalent/antibody, 1 micromole, in 1.0 mL DMSO) to 9 mL of this reoxidised antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO concentration. The solution was mixed for 1.5 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (4 micromoles, 40 μL at 100 mM), then diluted to >50 mL in PBS and sterile-filtered, ready for tangential flow filtration (TFF) purification.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjB at 280 nm and 330 nm (Compound 14 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 14, consistent with a drug-per-antibody ratio (DAR) of 1.88 molecules of Compound 14 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjB at 280 nm shows a monomer purity of 96%. UHPLC SEC analysis gives a concentration of final ConjB at 0.27 mg/mL in 50 mL, obtained mass of ConjB is 13.35 mg (89% yield).

Example 3

CB.17 SCID mice, aged 8-12 weeks, were injected with 1 mm³ BT474 tumour fragments sub cutaneously in the flank. When tumours reach an average size of 100-150 mm³, treatment is begun. Mice are weighed twice a week. Tumour size is measured twice a week. Animals are monitored individually. The endpoint of the experiment is a tumour volume of 1000 mm³ or 59 days, whichever comes first.

Groups of 10 xenografted mice were injected i.v. with 0.2 ml of antibody drug conjugate (ADC), in phosphate buffered saline (vehicle) or with 0.2 ml of vehicle alone. The concentration of ADC was adjusted to give 0.3 or 1.0 mg ADC/kg body weight in a single dose.

FIG. 1 shows the effect on mean tumour volume in groups of 10 mice dosed with ConjA at 0.3 or 1.0 mg/kg compared to vehicle control.

All regimens were acceptably tolerated with little body weight loss. The median time to end point (TTE) for vehicle-treated controls was 23.5 days, establishing a maximum possible tumour growth delay (TGD) of 35.5 days (151%) for the 59-day study. The ConjA regimen resulted in the maximum possible TGD, had ten of ten 59-day survivors and produced survival benefit that was statistically significantly different from vehicle-treated controls (P<0.001).

ConjA, at 1 mg/kg produced 100% regressions, nine partial regressions (PRs) and 1 complete regression (CR) which remained a tumour-free survivor (TFS) at study end.

ConjA at 0.3 mg/kg produced 60% regressions consisting of five PRs and one CR, that remained a TFS at study end.

Example 4—Toxicity Studies/Therapeutic Index

A single dose NonGLP toxicity study was used to determine the maximum tolerated dose (MTD) and safety profile of ConjA. Male Sprague Dawley rats (Harlan, Inc) were dosed once by slow bolus intravenous injection via the tail vein with vehicle control (25 mM Histidine-HCl, 7% sucrose, 0.02% Polysorbate 80, pH 6.0) or ConjA. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, clinical pathology (clinical chemistry, hematology, and coagulation), and gross pathology findings.

Animals that received 0.5 mg/kg of ConjA survived until scheduled necropsy on Study Day (SD) 29. Animals that received 1.0 mg/kg of ConjA were either found dead, or euthanized due to morbidity by SD 8.

Male Rats Dose Dose Main Study Group Treatment Route (mg/kg) Frequency N 1 Control IV 0 Single 5 3 ConjA IV 0.5 Single 5 6 ConjA IV 1.0 Single 5 Control/Vehicle for dilution = 25 mM Histidine-HCl, 7% sucrose, 0.02% Polysorbate 80, pH 6.0

Tolerability was determined based on toxicity end points, including body weight loss (>15%) and bone marrow suppression. Based on a lack of severe toxicity at 0.5 mg/kg, and mortality at 1.0 mg/kg, the maximum tolerated dose (MTD) in the rat after a single dose of ConjA was determined to be 0.5 mg/kg.

When compared to the minimum effective dose (MED) of 0.3 mg/kg in the BT474 xenograft model (see above), the potential therapeutic index (TI) for ConjA is 1.7 in rats, calculated as follows:

TI=MTD in rat (mg/kg)/MED in mouse efficacy model (mg/kg)

All documents and other references mentioned above are herein incorporated by reference. 

1. A compound with the formula I:

wherein: R² is of formula II′:

where A is a C₅₋₇ aryl group, X is selected from the group consisting of: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

and NHR^(N), wherein R^(N) is selected from H and C₁₋₄ alkyl, and either: (i) Q¹ is a single bond, and Q² is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or (ii) Q¹ is —CH═CH—, and Q² is a single bond; R¹² is selected from the group consisting of ═CH₂, ═CHR^(D1) and ═CR^(D1)R^(D2), where R^(D1) and R^(D2) are independently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹ are independently selected from the group consisting of: H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; R⁷ is selected from the group consisting of: H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Sn and halo; where R and R′ are independently selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups; either: (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl; (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R″ is a C₃₋₁₂ alkylene group, which chain is optionally interrupted by one or more heteroatoms, and/or aromatic rings; Y and Y′ are independently selected from the group consisting of O, S, and NH; R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ and R⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, wherein if R¹¹ and R^(11′) are SO_(z)M, M may represent a divalent pharmaceutically acceptable cation.
 2. A compound according to claim 1 wherein Y and Y′ are O, and R″ is unsubstituted C₃₋₇ alkylene. 3.-4. (canceled)
 5. A compound according to claim 1, wherein R⁶ and R⁹ are H.
 6. A compound according to claim 1, wherein R⁷ is selected from C₁₋₄ alkyoxy and benzyloxy.
 7. (canceled)
 8. A compound according to claim 1, wherein R² is phenyl.
 9. A compound according to claim 1, wherein X is NH₂. 10.-12. (canceled)
 13. A compound according to claim 1, wherein R¹² is ═CH₂. 14.-17. (canceled)
 18. A compound according to claim 1, which is of formula Ia:

where R^(1a) is selected from Me and Ph, and the amino group is at either the meta or para positions of the phenyl group. 19.-20. (canceled)
 21. A compound of formula II:

wherein R², R¹², R⁶, R⁷, R⁹, Y, R″, Y′, R^(6′), R^(7′), and R^(9′) are as defined in claim 1, and either: (a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹ is O-Prot^(O), wherein Prot^(O) is an oxygen protecting group; or (b) R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxo group. 22.-27. (canceled)
 28. A conjugate of formula (IIIa-1): L-(A ¹-L ¹-D)_(p)  (IIIa-1) wherein A¹ is of formula: -L ^(A)-A ² wherein L^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, and A² is selected from:

where n is 0 to 6;

where n is 0 to 6;

where n is 0 or 1, and m is 0 to 30;

where n is 0 or 1, and m is 0 to 30; where the asterisk indicates the point of attachment to L¹, the wavy line indicates the point of attachment to L^(A); and D is a Drug unit which is a PBD dimer according to claim 1, wherein L¹ is connected to D via the X substituent of R², and p is an integer of from 1 to
 20. 29. A conjugate of claim 28, wherein L^(A) is (L^(A-1)):

and A² is

where n is 1, and m is 0 to
 8. 30.-31. (canceled)
 32. A conjugate of claim 28, wherein L¹ is a dipeptide.
 33. The Conjugate of claim 32, wherein L¹ is selected from the group consisting of valine-alanine, valine-citrulline and phenyalanine-lysine. 34.-38. (canceled)
 39. A drug linker of formula IV: DLU-D  (IV) wherein DLU is a Drug Linker unit, and D is a Drug unit which is a PBD dimer according to any claim 1, wherein DLU is connected to D via the X substituent of R²; wherein DLU is of the formula: G ¹-L ¹-* wherein G¹ is of formula: G ¹-A ² where G^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, and A² is selected from:

where n is 0 to 6;

where n is 0 to 6;

where n is 0 or 1, and m is 0 to 30;

where n is 0 or 1, and m is 0 to 30; where the asterisk indicates the point of attachment to L¹, the wavy line indicates the point of attachment to G^(A).
 40. A drug linker of claim 39, wherein G^(A) is (G^(A-1)):

and A² is

where n is 1, and m is 0 to
 8. 41. A drug linker of claim 40, wherein m is
 8. 42. (canceled)
 43. A drug linker of claim 39, wherein L¹ is a dipeptide.
 44. A drug linker of claim 43, wherein L¹ is selected from the group consisting of valine-alanine, valine-citrulline and phenyalanine-lysine.
 45. A method of treatment of a proliferative disease comprising administering to a subject in need comprising administering to a subject in need of treatment a therapeutically effective amount of a compound according to claim
 1. 46. A method of treatment of a proliferative disease comprising administering to a subject in need comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate according to claim
 28. 